Injectable slurries and methods of manufacturing the same

ABSTRACT

A method for treating vascular diseases is provided. The method includes fabricating a sterile ice slurry including water and ice particles, cooling the sterile ice slurry to a predetermined temperature, and injecting the sterile ice slurry into a desired tissue region. The desired tissue region includes perivascular adipose tissue.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 15/505,042, which is a U.S. National Phase ofPCT/US2015/047301, filed on Aug. 27, 2015, which claims priority to U.S.Provisional Patent Application Ser. No. 62/042,979, filed Aug. 28, 2014,U.S. Provisional Patent Application Ser. No. 62/121,329, filed Feb. 26,2015, and U.S. Provisional Patent Application Ser. No. 62/121,472, filedFeb. 26, 2015. This application contains disclosure that is related toInternational Application No. PCT/US2015/047292, filed on Aug. 27, 2015.The present application also claims priority to U.S. Provisional PatentApplication Ser. No. 62/635,918, filed on Feb. 27, 2018. The entiredisclosures of the aforementioned applications are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

Although various devices and techniques seek to selectively targetcertain tissues, there remains a need for higher resolution andspecificity in targeting selected tissues while avoiding harm tonon-selected tissues.

SUMMARY OF THE INVENTION

One aspect of the invention provides a slurry comprising: a plurality ofsterile ice particles having a largest cross-sectional dimension lessthan about 1.5 mm; and a biocompatible surfactant. In one aspect, thepresent disclosure provides

This aspect of the invention can have a variety of embodiments. Theplurality of sterile ice particles can have a largest cross-sectionaldimension less than a value selected from the group consisting of: about1.25 mm, about 1 mm, about 0.9 mm, about 0.8 mm, about 0.7 mm, about 0.6mm, about 0.5 mm, about 0.4 mm, about 0.3 mm, about 0.2 mm, and about0.1 mm.

The biocompatible surfactant can be one or more selected from the groupconsisting of: a solvent, a detergent, a wetting agent, an emulsifier, afoaming agent, and a dispersant. The biocompatible surfactant can be oneor more selected from the group consisting of: anionic, cationic,amphoteric, and nonionic. The biocompatible surfactant can be glycerol.The biocompatible surfactant can be urea. The foregoing and otheraspects and advantages of the disclosure will appear from the followingdescription. In the description, reference is made to the accompanyingdrawings which form a part hereof, and in which there is shown by way ofillustration a preferred configuration of the disclosure. Suchconfiguration does not necessarily represent the full scope of thedisclosure, however, and reference is made therefore to the claims andherein for interpreting the scope of the disclosure.

The plurality of ice particles can constitute between about 0.1% andabout 75% of the slurry by weight. The plurality of ice particles canconstitute a percentage by weight selected from the group consisting of:between about 0.1% and 1%, between about 1% and 10%, between about 10%and about 20%, between about 20% and about 30%, between about 30% andabout 40%, between about 40% and about 50%, between about 50% and about60%, between about 60% and about 70%, and greater than about 50%. Theplurality of ice particles can constitute between about 0.1% and about50% of the slurry by weight.

The slurry can further include a therapeutic compound.

The therapeutic compound can be selected from the group consisting of:an anesthetic and an analgesic. The therapeutic compound can be awater-soluble anesthetic. The therapeutic compound can be selected fromthe group consisting of: prilocaine, bupivacaine, prilocaine,tetracaine, procaine, mepivicaine, etidocaine, lidocaine, QX-314, and anonsteroidal anti-inflammatory drugs (NSAID).

The therapeutic compound can be a vasoconstrictor. The vasoconstrictorcan be selected from the group consisting of: epinephrine,norepinephrine, a selective adrenergic agonist, a nonselectiveadrenergic agonist, and a corticosteroid.

The slurry can further include one or more selected from the groupconsisting of: microbubbles, nanobubbles, and biodegradable solids.

The slurry can further include a toxin. The toxin can be ethanol.

The slurry can be hypertonic. The slurry can be hypotonic.

The slurry can have a mean temperature selected from the groupconsisting of: about +10° C., about +9° C., about +8° C., about +7° C.,about +6° C., about +5° C., about +4° C., about +3° C., about +2° C.,about +1° C., about 0° C., about −1° C., about 2° C., about −3° C.,about −4° C., about 5° C., about −6° C., about 7° C., about 8° C., about9° C., about 10° C., about 11° C., about 12° C., about 13° C., about 14°C., about 15° C., between about 15° C. and about 25° C., between about25° C. and about 50° C., and between about 50° C. and about 75° C.

The slurry can have a mean temperature of about +5° C. or lower.

Another aspect of the invention provides a slurry including: a pluralityof sterile ice particles having a largest cross-sectional dimension lessthan about 1.5 mm; a biocompatible surfactant; and a foam comprising aplurality of gas bubbles.

This aspect of the invention can have a variety of embodiments. Theslurry can have a mean temperature selected from the group consistingof: about +10° C., about +9° C., about +8° C., about +7° C., about +6°C., about +5° C., about +4° C., about +3° C., about +2° C., about +1°C., about 0° C., about −1° C., about 2° C., about −3° C., about −4° C.,about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., about10° C., about 11° C., about 12° C., about 13° C., about 14° C., about15° C., between about 15° C. and about 25° C., between about 25° C. andabout 50° C., and between about 50° C. and about 75° C.

Another aspect of the invention provides a slurry including: a pluralityof sterile ice particles having a largest cross-sectional dimension lessthan about 1.5 mm; and a biocompatible excipient.

This aspect of the invention can have a variety of embodiments. Theslurry can have a mean temperature selected from the group consistingof: about +10° C., about +9° C., about +8° C., about +7° C., about +6°C., about +5° C., about +4° C., about +3° C., about +2° C., about +1°C., about 0° C., about −1° C., about 2° C., about −3° C., about −4° C.,about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., about10° C., about 11° C., about 12° C., about 13° C., about 14° C., about15° C., between about 15° C. and about 25° C., between about 25° C. andabout 50° C., and between about 50° C. and about 75° C.

Another aspect of the invention provides a slurry including: a pluralityof sterile ice particles having a largest cross-sectional dimension lessthan about 1.5 mm; and a lipolytic agent.

This aspect of the invention can have a variety of embodiments. Thelipolytic agent can be a detergent. The detergent can be deoxycholate.The lipolytic agent can be an alcohol. The lipolytic agent can be anorganic solvent.

The slurry can have a mean temperature selected from the groupconsisting of: about +10° C., about +9° C., about +8° C., about +7° C.,about +6° C., about +5° C., about +4° C., about +3° C., about +2° C.,about +1° C., about 0° C., about −1° C., about 2° C., about −3° C.,about −4° C., about 5° C., about −6° C., about 7° C., about 8° C., about9° C., about 10° C., about 11° C., about 12° C., about 13° C., about 14°C., about 15° C., between about 15° C. and about 25° C., between about25° C. and about 50° C., and between about 50° C. and about 75° C.

Another aspect of the invention a method of treating a subject. Themethod includes: injecting a slurry as described herein into a treatmentregion of the subject.

This aspect of the invention can have a variety of embodiments. Thetreatment region can be selected from the group consisting of: proximateto a nerve, proximate to subcutaneous adipose tissue, proximate tobreast tissue, proximate to visceral fat, fatty tissue proximate to thepharynx, fatty tissue proximate to the palate, fatty tissue proximate tothe tongue, proximate to a spinal cord lipoma, proximate to alipomyelomeningocele, proximate to visceral fat, proximate tolipomastia, proximate to a tumor, proximate to cardiac tissue, proximateto pericardial fat, and proximate to epicardial fat.

The treatment region can include one or more tissues selected from thegroup consisting of: connective, epithelial, neural, joint, cardiac,adipose, hepatic, renal, vascular, cutaneous, and muscle.

The method can further include measuring a temperature of the slurryprior to injection.

The slurry can be injected via gravity flow. The slurry can be injectedvia pressure injection. The slurry can be injected through one or moreselected from the group consisting of: a syringe, a cannula, a catheter,and tubing.

The method can further include pre-cooling the treatment region prior tothe injecting step. The method can further include applying energyadjacent to the target tissue. The method can further include applyingsuction to the treatment region to remove melted slurry.

The injecting step can include injecting a sufficient volume of theslurry to cause tumescent swelling of the treatment region. Theinjecting step can be repeated a plurality of times.

The method can further include calculating a desired amount of slurry tobe injected based on a desired amount of disruption to the treatmentregion. The slurry can cool the treatment region adjacent to aninjection site at a rate greater in magnitude than about −2° C. perminute.

The method can thicken septa. The septa can be in adipose tissue and/ordermis.

The method can further include mixing the slurry with relatively warmerliquid prior to the injecting step.

The slurry can have a mean temperature selected from the groupconsisting of: about +10° C., about +9° C., about +8° C., about +7° C.,about +6° C., about +5° C., about +4° C., about +3° C., about +2° C.,about +1° C., about 0° C., about −1° C., about 2° C., about −3° C.,about −4° C., about 5° C., about −6° C., about 7° C., about 8° C., about9° C., about 10° C., about 11° C., about 12° C., about 13° C., about 14°C., about 15° C., between about 15° C. and about 25° C., between about25° C. and about 50° C., and between about 50° C. and about 75° C.

The slurry can be hypotonic relative to the treatment region. The slurrycan be isotonic relative to the treatment region. The slurry can behypertonic relative to the treatment region.

Another aspect of the invention provides a method of treating a subject.The method includes: injecting the slurry as described herein having atemperature and cooling capacity sufficient to non-selectively disrupttissue into a treatment region of the subject.

This aspect of the invention can have a variety of embodiments. Thetreatment region can be selected from the group consisting of: aprostate, a kidney, a heart, and a fibroadenoma.

The slurry can have a mean temperature selected from the groupconsisting of: about +10° C., about +9° C., about +8° C., about +7° C.,about +6° C., about +5° C., about +4° C., about +3° C., about +2° C.,about +1° C., about 0° C., about −1° C., about 2° C., about −3° C.,about −4° C., about 5° C., about −6° C., about 7° C., about 8° C., about9° C., about 10° C., about 11° C., about 12° C., about 13° C., about 14°C., about 15° C., between about 15° C. and about 25° C., between about25° C. and about 50° C., and between about 50° C. and about 75° C.

The slurry can be hypotonic relative to the treatment region. The slurrycan be isotonic relative to the treatment region. The slurry can behypertonic relative to the treatment region.

Another aspect of the invention provides a method of treating a subject.The method includes injecting a slurry into a treatment region of thesubject selected from the group consisting of: proximate to a nerve,proximate to subcutaneous adipose tissue, proximate to breast tissue,proximate to visceral fat, fatty tissue proximate to the pharynx, fattytissue proximate to the palate, fatty tissue proximate to the tongue,proximate to a spinal cord lipoma, proximate to a lipomyelomeningocele,proximate to visceral fat, proximate to lipomastia, proximate to atumor, proximate to cardiac tissue, proximate to pericardial fat, andproximate to epicardial fat.

This aspect of the invention can have a variety of embodiments. Theslurry can include an ionic component.

The slurry can have a temperature and cooling capacity sufficient tonon-selectively disrupt tissue into a treatment region of the subject.The slurry can have a mean temperature selected from the groupconsisting of: about +10° C., about +9° C., about +8° C., about +7° C.,about +6° C., about +5° C., about +4° C., about +3° C., about +2° C.,about +1° C., about 0° C., about −1° C., about 2° C., about −3° C.,about −4° C., about 5° C., about −6° C., about 7° C., about 8° C., about9° C., about 10° C., about 11° C., about 12° C., about 13° C., about 14°C., about 15° C., between about 15° C. and about 25° C., between about25° C. and about 50° C., and between about 50° C. and about 75° C.

Another aspect of the invention provides a method of preparing a slurry.The method includes: freezing a plurality of sterile ice particleshaving a largest cross-sectional dimension less than about 1.5 mm in oneor more micromolds; and mixing the plurality of sterile ice particleswith one or more biocompatible liquids.

This aspect of the invention can have a variety of embodiments. Theplurality of sterile ice particles can have a substantially uniformshape. The one or more micromolds can be fabricated from one or morematerials selected from the group consisting of: polymers, plastics,elastomers, silicons, silicones, and metals. The method can furtherinclude applying mechanical strain, stress waves, shock waves, orcentripetal force to remove the plurality of sterile ice particles fromthe one or more micromolds.

The slurry can have a mean temperature selected from the groupconsisting of: about +10° C., about +9° C., about +8° C., about +7° C.,about +6° C., about +5° C., about +4° C., about +3° C., about +2° C.,about +1° C., about 0° C., about −1° C., about 2° C., about −3° C.,about −4° C., about 5° C., about −6° C., about 7° C., about 8° C., about9° C., about 10° C., about 11° C., about 12° C., about 13° C., about 14°C., about 15° C., between about 15° C. and about 25° C., between about25° C. and about 50° C., and between about 50° C. and about 75° C.

Another aspect of the invention provides a slurry comprising: aplurality of sterile ice particles having a largest cross-sectionaldimension less than about 1.5 mm; and an ionic component selected fromthe group consisting of: hydrogen ions, lactate, phosphate, zinc ions,sulfur ions, nitrate, ammonium, hydroxide, iron ions, barium ions.

In some aspects, the slurries disclosed herein may be used to treatvascular disease. In some aspects, the present disclosure provides amethod for treating vascular disease that includes fabricating a sterileice slurry including water and ice particles, cooling the sterile iceslurry to a predetermined temperature, and injecting the sterile iceslurry into a desired tissue region. The desired tissue region includesperivascular adipose tissue.

In some aspects, the present disclosure provides an injection systemconfigured to access perivascular adipose tissue. The injection systemincludes a sterile ice slurry including water and ice particles. Thesterile ice slurry is at a predetermined temperature. The injectionsystem further includes an injection device configured to inject thesterile ice slurry into or around perivascular adipose tissue. The iceparticles in the sterile ice slurry define a largest cross-sectionaldiameter of less than 2 millimeters to enable the sterile ice slurry toflow through the injection device.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the nature and desired objects of thepresent invention, reference is made to the following detaileddescription taken in conjunction with the accompanying drawing figureswherein like reference characters denote corresponding parts throughoutthe several views.

FIG. 1 depicts a method of preparing a slurry according to an embodimentof the invention.

FIG. 2 depicts a method of preparing a slurry according to an embodimentof the invention.

FIG. 3 depicts an experimental prototype for generating slurriesaccording to an embodiment of the invention.

FIG. 4 depicts an example of ice produced by introducing dropletsgenerated by an ultrasonic humidifier into a dry ice environmentaccording to an embodiment of the invention.

FIG. 5 depicts ice balls harvested using liquid nitrogen according to anembodiment of the invention.

FIG. 6 depicts a general method of treatment using injectable slurriesaccording to an embodiment of the invention.

FIG. 7 illustrates an injection system configured to inject slurry intoor around a desired tissue region.

FIG. 8 illustrates the injection system of FIG. 7 including a suctiondevice.

FIG. 9 illustrates an injection system configured to inject slurry intoor around a desired tissue region that includes a balloon-basedcatheter.

FIG. 10 depicts an ice slurry with a high concentration of small iceparticles according to an embodiment of the invention.

FIGS. 11A, 11B and 11C depict the results of injection of an ice slurryinto human abdominoplasty adipose tissue according to an embodiment ofthe invention.

FIGS. 12A and 12B depict the detection of injected slurry withultrasound in ex vivo human skin according to an embodiment of theinvention. FIG. 12A is an ultrasound image of human skin prior to slurryinjection. FIG. 12B is an ultrasound image of human skin after slurryinjection.

FIG. 13 depicts a chart of the temperature of an ex vivo humanabdominoplasty specimen being heated from below after injection of acold slurry according to an embodiment of the invention.

FIGS. 14A and 14B depict gross photographs of pig skin 4 weeks afterinjections of a melted, room temperature slurry (FIG. 14A) and coldslurry (FIG. 14B) according to embodiments of the invention.

FIGS. 15A and 15B depict ultrasound images of pig skin at a treatmentsite prior to cold slurry injection (FIG. 15A) and 4 weeks after coldslurry injection (FIG. 15B) according to an embodiment of the invention.

FIGS. 16A and 16B depict gross photographs of pig skin at anothertreatment site 4 weeks after injections of cold slurry according to anembodiment of the invention demonstrating a marked depression in skincaused by loss of subcutaneous fat at the site of the injection.

FIGS. 17A and 17B depict ultrasound images of pig skin at anothertreatment site prior to cold slurry injection (FIG. 17A) and 4 weeksafter cold slurry injection (FIG. 17B) according to an embodiment of theinvention.

FIG. 18 depicts the creation of a slurry using a benchtop analyticalmill according to an embodiment of the invention.

FIG. 19 depicts an injection site in the area of the left inguinal fatpad in adult Sprague-Dawley rats.

FIGS. 20A, 20B, and 20C depicts the result of injection of roomtemperature hetastarch solutions, injection of cold hetastarch slurry,and no injection in a control site, respectively, in adultSprague-Dawley rats.

FIGS. 20D, 20E, 20F, and 20G depict tissue surrounding the injectionsite demonstrating no effects on muscle or surrounding tissue.

FIGS. 21A and 21B depict the result of injections of 5% TWEEN® 20(polysorbate 20) in lactated Ringer's solution plus 5% dextrose at roomtemperature (+16° C.) and cold slurry (0.6° C.), respectively, in adultSprague-Dawley rats.

FIG. 21C depicts the control (not injected) side.

FIGS. 21D, 21E, 21F, and 21G depict tissue surrounding the injectionsite demonstrating no effect on muscle or surrounding tissue.

FIGS. 22A and 22B depict the result of injections of 5% polyethyleneglycol (PEG) in lactated Ringer's solution plus 5% dextrose at roomtemperature (+8° C.) and cold slurry (0.8° C.), respectively, in adultSprague-Dawley rats.

FIG. 22C depicts the control (not injected) side.

FIGS. 22D, 22E, 22F, and 22G depict tissue surrounding the injectionsite demonstrating no effect on muscle or surrounding tissue.

FIG. 23A depicts injection sites on a swine before injection.

FIG. 23B depicts injection sites 14 days after injection.

FIG. 24 depicts a graph of cooling at three locations during slurryinjection into a swine.

FIGS. 25A-25D are photographs of injection site 11, which received aninjection of normal slurry with 10% glycerol at −4.1° C.

FIGS. 26A and 26B depict a foamy slurry.

FIG. 27 depicts the use of a foamy slurry as an insulator for furtherslurry injection(s).

FIG. 28A depicts an injection site for porcine parapharyngeal and neckfat pads. FIG. 28B depicts the injection depth. FIGS. 28C and 28D depictthe localization of the slurry (containing ink) within theparapharyngeal and neck fat pads.

FIGS. 29A-29K are photographs depicting the result of various slurrycompositions into swine.

FIGS. 30A, 30B, and 30C depict various structures for removal of meltedslurry from an injection site according to an embodiment of theinvention.

FIG. 31 depicts a tray for molding micro ice particles and a micro iceparticle that can be formed from such a tray according to an embodimentof the invention.

FIGS. 32A, 32B, 32C, and 32D depict histology of the perigonadalvisceral fat of obese mice.

FIG. 33 is a graph of average weight loss of obese mice treated withintraperitoneal injection of cold slurry compared to their untreatedcohort.

FIGS. 34A and 34B provides images of gross biopsies of swine taken attime of sacrifice three months post-procedure and showing a visibledermal thickening in the treated region.

FIGS. 35A and 35B provide images of histology of swine taken at time ofsacrifice three months post-procedure and stained with hematoxylin andeosin (H&E).

FIG. 36A depicts a quantitative model to illustrate the behavior ofinjected slurries according to an embodiment of the invention.

FIG. 36B depicts three stages of heat exchange following infusion of aslurry into a tissue.

FIGS. 37A and 37B provide images of immunohistochemical (IHC) stainingfor type I collagen taken at time of sacrifice three monthspost-procedure.

FIGS. 38A and 38B provide images of immunohistochemical (IHC) stainingfor type III collagen taken at time of sacrifice three monthspost-procedure.

FIGS. 39A and 39B are magnetic resonance (MR) images depicting thecross-sections of a control mouse trachea and adjacent tissue at abaseline and four week follow-up, respectively.

FIGS. 40A and 40B are magnetic resonance (MR) images depicting thecross-sections of a treated mouse trachea and adjacent tissue at abaseline and four week follow-up, respectively.

FIG. 41 illustrates a slurry injection into epicardial fat aroundcoronary arteries and surrounding a beating swine heart.

FIG. 42 is a graph illustrating temperature in swine pericardial fat,swine epicardial fat, and swine thoracic peri-aortic fat as a functionof time following a slurry injection.

FIG. 43 is a graph illustrating temperature in swine pericardial fat asa function of time following a slurry injection.

FIG. 44 illustrates a slurry injection into swine perivascular adiposetissue following a left thoracotomy.

FIG. 45 is a graph illustrating percent change in adipose tissuethickness following slurry injections with varying ice contents andvolumes.

FIG. 46 is a graph illustrating a total volume loss of adipose tissuefollowing slurry injections with varying ice contents and volumes.

DEFINITIONS

The instant invention is most clearly understood with reference to thefollowing definitions.

As used herein, the singular form “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. “About” canbe understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

As used in the specification and claims, the terms “comprises,”“comprising,” “containing,” “having,” and the like can have the meaningascribed to them in U.S. patent law and can mean “includes,”“including,” and the like.

Unless specifically stated or obvious from context, the term “or,” asused herein, is understood to be inclusive.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (aswell as fractions thereof unless the context clearly dictatesotherwise).

As used herein, the term “slurry” refers to a plurality of ice particlesin an aqueous solution.

DETAILED DESCRIPTION OF THE INVENTION

Various aspects of the invention involve introducing a compositionincluding a cold slurry (e.g., ice slurry) into tissue, e.g., directlyinto the tissue rather than through a natural conduit of the body suchas arteries, veins, or gut. When a volume of ice slurry is directlyintroduced into a volume of soft tissue, there is rapid heat exchangebetween the tissue and the slurry. When rapidly and locally injected, apool of slurry is produced that contacts a target volume of localtissue. By contrast, if a slurry is infused more slowly and with largervolume, the slurry penetrates and flows through spaces in the tissue,producing widespread channels filled with slurry in a process similar tothe administration of tumescent anesthesia. Infusion enables sustainedflow of slurry through tissue, especially tissue nearby the site ofintroduction. This tissue can be profoundly cooled to the temperature ofthe slurry itself by the continuous or prolonged flow of slurry.

In general, there are two periods of heat exchange upon injection ofslurry directly into tissue: a rapid equilibration between slurry andlocal tissue, followed by slower warming to body temperature. During therapid equilibration, the slurry is warmed and the local tissue iscooled, until an equilibrium temperature is reached that is between theinitial temperatures of the slurry and the tissue. During this rapidtissue cooling by heat exchange, three events occur: (1) heat stored bythe heat capacity of the slurry and the tissue is exchanged; (2) heatreleased by the crystallization of tissue lipids is exchanged; and (3)heat absorbed by melting of slurry ice is exchanged. Some or all of theice in the slurry melts, and some or all of the lipids in the tissue arecrystallized, according to the parameters of the tissue and the slurry.

After the rapid heat exchange with the slurry, there is gradual warmingby heat exchange with the body. Gradual warming occurs by a combinationof heat diffusion from surrounding warm tissue and by convective heatingfrom blood flow. Blood flow can be reduced in the local tissue bypressure or by drugs as discussed in greater detail herein. The desiredlevel of pain relief may depend on temperature, rate of cooling,duration of cooling and the number of cooling cycles.

Injectable Slurries

Embodiments of the invention provide injectable slurries that can beused for selective or non-selective cryotherapy and/or cryolysis.Without being bound by theory, it is believed that such slurries cantarget and disrupt desired tissue through the extraction of heat fromadjacent tissue during melting of the ice component of the slurry.

In addition, the osmolality or osmolarity of the slurry can be adjustedto synergistically induce selective damage through hypertonic orhypotonic injury. For example, the slurries can be isotonic slurrieshaving an osmolarity of about 308 mOsm/L, hypotonic slurries having anosmolarity less than about 308 mOsm/L, or hypertonic slurries having anosmolarity greater than about 308 mOsm/L.

As discussed herein, varying amounts of additives such as freezing pointdepressants can be added to the slurries. For example, the additives canconstitute less than about 20% w/w of the slurry, between about 20% andabout 40% w/w of the slurry, and the like.

In one embodiment, the injectable slurry includes a plurality of sterileice particles and one or more freezing point depressants. The freezingpoint depressants can also alter the viscosity of the slurry, preventagglomeration of the ice particles, increase thermal conductivity offluid phase, and otherwise improve the performance of the slurry.

The degree of freezing point depression can be calculated either usingthe idealized formula:ΔT _(F) =K _(F) bi

wherein ΔT_(F) is the freezing point depression (as defined byT_(F(pure solvent))−T_(F(solution))), K_(F) is the cryoscopic constant,b is molality, and i is the van 't Hoff factor representing the numberof ion particles per individual molecule of solute (e.g., 2 for NaCl, 3for BaCl₂) or in the formulas proposed in X. Ge & X. Wang, “Estimationof Freezing Point Depression, Boiling Point Elevation and Vaporizationenthalpies of electrolyte solutions,” 48 Ind. Eng. Chem. Res. 2229 35(2009) and X. Ge & X. Wang, “Calculations of Freezing Point Depression,Boiling Point Elevation, Vapor Pressure and Enthalpies of Vaporizationof Electrolyte Solutions by a Modified Three-Characteristic ParameterCorrelation Model,” 38 J. Sol. Chem. 1097-1117 (2009).

In order to ensure that the slurry can be injected into a subjectthrough a needle, a cannula, or a catheter, the size of the iceparticles can be controlled. Without being bound by theory, it isbelieved that a slurry will be injectable if all or most (e.g., greaterthan about 50% by quantity, greater than about 75% by quantity, greaterthan about 80% by quantity, greater than about 90% by quantity, greaterthan about 95% by quantity, greater than about 99% by quantity, and thelike) of the ice particles have a largest cross-sectional dimension(i.e., the largest distance between any two points on the surface of theice particle) no greater than half of the internal diameter of thevessels (e.g., needles, cannulae, catheters, tubing, and the like) to beused. For example, if the slurry is to be injected using a catheterhaving a 3 mm internal diameter, the ice particles will preferably havea largest cross-sectional dimension less than or equal to about 1.5 mm.In some embodiments, the ice particles have a mean largestcross-sectional dimension of 1 mm or less.

As discussed in greater detail herein, this controlled size can beachieved by controlled generation or processing of the ice particlesand/or by filtering, screening, or sorting of the ice particles.Controlled storage, transport, and/or handling of the ice particlesand/or slurries can also promote predictable, flowable slurries bypreventing thawing and refreezing of the ice particles, which may changethe size of the ice particles and/or produce sharp and/or jaggedsurfaces.

Without being bound by theory, exemplary suitable ice particle sizes forvarious internal catheter diameters and internal needle diameters areprovided in Table 1 and Table 2, respectively, below.

TABLE 1 Recommended Largest Ice Particle Cross-Sections by Catheter SizeRecommended Largest Cross- Catheter Internal Diameter Section of IceParticles 4 mm 2 mm 3 mm 1.5 mm 2 mm 1 mm

TABLE 2 Recommended Largest Ice Particle Cross-Sections by Needle SizeNeedle Recommended Largest Cross- Gauge Nominal Internal DiameterSection of Ice Particles  7 3.81 mm 1.905 mm  8 3.429 mm 1.7145 mm  92.997 mm 1.4985 mm 10 2.692 mm 1.346 mm 11 2.388 mm 1.194 mm 12 2.159 mm1.0795 mm 13 1.803 mm 0.9015 mm 14 1.6 mm 0.8 mm 15 1.372 mm 0.686 mm 161.194 mm 0.597 mm 17 1.067 mm 0.5335 mm 18 0.838 mm 0.419 mm 19 0.686 mm0.343 mm 20 0.603 mm 0.3015 mm 21 0.514 mm 0.257 mm 22 0.413 mm 0.2065mm  22s 0.152 mm 0.076 mm 23 0.337 mm 0.1685 mm 24 0.311 mm 0.1555 mm 250.26 mm 0.13 mm 26 0.26 mm 0.13 mm  26s 0.127 mm 0.0635 mm 27 0.21 mm0.105 mm 28 0.184 mm 0.092 mm 29 0.184 mm 0.092 mm 30 0.159 mm 0.0795 mm31 0.133 mm 0.0665 mm 32 0.108 mm 0.054 mm 33 0.108 mm 0.054 mm 340.0826 mm 0.0413 mm

One or more freezing point depressants can be added to form sub-0° C.slurries that remain injectable. Freezing point depressants can alsoreduce the temperature of the slurries to temperatures below 0□ C.Suitable freezing point depressants include biocompatible compounds suchsalts (e.g., sodium chloride), ions, Lactated Ringer's solution, sugars(e.g., glucose, sorbitol, mannitol, hetastarch, sucrose, or acombination thereof), biocompatible surfactants such as glycerol (alsoknown as glycerin or glycerine), other polyols, other sugar alcohols,and/or urea, and the like. In particular, some biocompatible surfactantssuch as glycerol are believed to cause ice particles to shrink andbecome rounder and also serves as a cryo-protectant for non-lipid-richcells. Other exemplary biocompatible surfactants include sorbitan estersof fatty acids, polysorbates, polyoxyethylene sorbitan monooleate (alsoknown as polysorbate 80 and available under the TWEEN® 80 trademark fromCroda Americas LLC of New Castle, Del.), sorbitan monooleatepolyoxyethylene sorbitan monolaurate (also known as polysorbate 80 andavailable under the TWEEN® 80 trademark from Croda Americas LLC of NewCastle, Del.), polysorbate 20 (polyoxyethylene (20) sorbitanmonolaurate), polysorbate 40 (polyoxyethylene (20) sorbitanmonopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitanmonostearate), polysorbate 80 (polyoxyethylene (20) sorbitanmonooleate), sorbitan ester, poloxamater, lecithin,polyoxyethylene-polyoxypropylene copolymers (available under thePLURONICS® trademark from BASF Corporation of Mount Olive, N.J.),sorbitan trioleate (available under the SPAN® 85 trademark fromSigma-Aldrich of St. Louis, Mo.) and the like.

Surfactants can also act as a solvent, detergent, wetting agent,emulsifier, foaming agent, and/or dispersant. Surfactants can beanionic, cationic, amphoteric, or nonionic. Biocompatible surfactantscan be included in injectable ice slurries.

Injectable slurries can be configured to have a desired temperature andto extract a desired amount of heat per unit of volume or mass ofslurry. Specifically, the solute (i.e., freezing point depressant)concentration dictates the temperature of the slurry and the ice contentof the slurry determines the amount of heat extracted by the slurry.

For selectively-destructive slurries that target the relativevulnerability of lipid-rich cells, the slurry is preferably isotonicrelative to the subject's cells, e.g., having an osmolarity of about 308mOsm/L. For example, slurries including normal saline and 20% glycerolwere able to target lipid rich cells while avoiding acute unselectivenecrosis. Broadly destructive slurries can achieve colder temperaturesand greater destructive power by increasing the solute concentration(e.g., to 20% w/v saline) to form a hypertonic solution (i.e., asolution having an osmolarity greater than about 308 mOsm/L) that willalso disrupt cells through osmotic pressure. As the ice melts, thesolute concentration will decrease.

The injectable slurries can contain varying proportions of ice. Forexample, the slurries can contain between about 0.1% and about 75% iceby weight, between about 0.1% and 1% ice by weight, between about 1% and10% ice by weight, between about 10% and about 20% ice by weight,between about 20% and about 30% ice by weight, between about 30% andabout 40% ice by weight, between about 40% and about 50% ice by weight,between about 50% and about 60% ice by weight, between about 60% andabout 70% ice by weight, and greater than about 50% ice by weight. (Theproportions of ice by volume are slightly higher due to the densities ofsolid and liquid water.)

In some embodiments, the injectable slurry further comprises atherapeutic compound (which can be included in calculating the soluteconcentration above). The therapeutic compound can be a liquid, a gas,or a solid.

In one embodiment, the therapeutic compound is an anesthetic and/or ananalgesic, for example, a water-soluble anesthetic (e.g., prilocaine),bupivacaine, prilocaine, tetracaine, procaine, mepivicaine, etidocaine,lidocaine, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids(e.g., methylprednisone), and the like. Inclusion of an anesthetic inthe slurry can be particularly advantageous when the slurry is used toprovide a nerve block because (i) the effect of the anesthetic willprovide immediate confirmation that the slurry is being injected in thecorrect location and (ii) the anesthetic can provide pain relief untilthe cyroneurolysis nerve block is effective (potentially after 48hours).

In one embodiment, the anesthetic is QX-314, N-ethyl bromide, aquaternary lidocaine derivative that is a permanently charged moleculecapable of providing long term (over 24 hours) anesthesia. Unlikelidocaine, QX-314 can provide more selective blocking of nociceptors andwith longer duration of action and less side effects. QX-314 is acharged molecule that needs to enter the cell and block the sodiumchannels intracellularly. The ability of QX-314 to block from theinside, but not the outside of neuronal membranes could be exploited toblock only desired neurons. Combining QX-314 with the cold slurryinjections described herein can selectively target cold sensingnociceptive sensory neurons to provide selective and long lastinganesthesia.

In another embodiment, the therapeutic compound is a vasoconstrictorsuch as epinephrine, norepinephrine, other selective or nonselectiveadrenergic agonists, or a corticosteroid. Vasoconstrictors canadvantageously prolong the cooling effect of the slurry by reducingwarming from blood flow. (Pressure or suction can also be used to reduceblood flow and/or isolate the target tissue as discussed in U.S. Pat.Nos. 7,367,341 and 8,840,608.)

In still another embodiment, the injectable slurry can include one ormore lipolytic agents to enhance the reduction of lipid-rich cells.Exemplary lipolytic agents include biocompatible surfactants, bile saltsand their derivatives (e.g., deoxycholic acid), phosphatidylcholine(lecithin), catecholamines, B-agonists (e.g., isoproterenol), alpha2-agonists (e.g., yohimbine), phosphodiesterase inhibitors (e.g.,aminophylline, theophylline), corticosteroids, caffeine, hyalorunidase,collagenase, alpha-tocopherol, ethanol, benzyl alcohol, carnitine,catechin, cysteine, gallic acid, laminarin, rutin, myrecetin, alpha MSH,melilotus, resveratrol, genistein, and the like. Such agents can disruptadipose tissue morphology when injected at room temperature and beparticularly useful in augmenting disruption of adipose tissuemorphology when included in slurries prepared for cryolipolysis and/orcryoneurolysis. Table 3 provides a list of exemplary lipolytic agents,cell targets, and hypothesized mechanisms of action.

TABLE 3 Exemplary Lipolytic (Lipid-Rich-Cell-Targeting) Agents Cell CellCompound Category Target Lysis? Hypothesized Mechanism of Action Bilesalts and their derivatives Adipocyte Yes Solubilize/break downadipocyte cell (e.g., deoxycholic acid) membrane through detergenteffect Phosphatidylcholine (lecithin) Adipocyte No Buffers ablativeeffects of detergents (e.g., deoxycholate, cholates, and the like)Catecholamines, B-agonists Adipocyte No Increased lipolysis; stimulationof B- (e.g., isoproterenol) adrenoreceptor increases cAMP Alpha2-agonists (e.g., yohimbine) Adipocyte No Increased lipolysisPhosphodiesterase inhibitors (e.g., Adipocyte No Increased lipolysis;phosphodiesterase aminophylline, theophylline) inhibition increases cAMPCorticosteroids Adipocyte No Block downregulation of B-adrenoreceptorsForskolin (plant derivative: Adipocyte No Increased lipolysis; activatesadenylate Coleus forskohlii) cyclase independent of adrenoreceptorresulting in increased cAMP Caffeine Adipocyte No Increased lipolysis;multiple mechanisms (Phosphodiesterase inhibition, increasedcatecholamine release, adenosine antagonist) Collagenase Fibrous N/ADissolves fibrous septae septae

In some embodiments, the injectable slurry further includes microbubblesor nanobubbles to aid in imaging (particularly by ultrasound) andverification of the injection site. Suitable microbubbles andnanobubbles and methods for making the same are described in U.S. Pat.Nos. 7,897,141 and 8,715,622 and U.S. Patent Application Publication No.2008/0247957, 2008/0279783, and 2009/0028797.

In some embodiments, specifically non-selective injectable slurries, theslurry can further include a toxin or sclerosing agents such as ethanol,detergents, and the like.

Slurries can contain other emulsifiers and excipients included in otherparenteral solutions such as those described in Sougata Pramanick etal., “Excipient Selection In Parenteral Formulation Development,” 45(3)Pharma Times 65-77 (2013). Exemplary excipients are listed in Table 4below. The substances described herein can be administered in a varietyof doses that can produce varying effects. For example, low doses of aparticular substance can act as an inert excipient, but exert atherapeutic effect at a higher concentration.

TABLE 4 Exemplary Excipients Category Examples Bulking Agents Sucrose,lactose, trehalose, mannitol, sorbitol, glucose, raffinose, glycine,histidine, PVP (K40) Buffering Agents Sodium citrate, sodium phosphate,sodium hydroxide, tris base-65, tris acetate, tris HCl- 65 TonicityDextrose Modifiers Collapse Dextran, ficoll, gelatin, hydroxyethylstarch Temperature Modifiers Antimicrobial Benzalkonium chloride,benzethonium chloride, benzyl alcohol, Preservatives chlorobutanol,m-cresol, myristyl gamma-picolinium chloride, paraben methyl, parabenpropyl, phenol, 2-penoxyethanol, phenyl mercuric nitrate, thimerosalChelating Agents Calcium disodium EDTA (ethylenediaminetetra aceticacid), disodium EDTA, calcium versetamide Na, calteridol, DTPAAntioxidant and Acetone sodium bisulfate, argon, ascorbyl palmitate,ascorbate Reducing Agents (sodium/acid), bisulfite sodium, butylatedhydroxyl anisole, butylated hydroxyl toluene (BHT), cystein/cysteinateHCl, dithionite sodium, gentistic acid, gentistic acid ethanolamine,glutamate monosodium, glutathione, formaldehyde sulfoxylate sodium,metabisulfite potassium, metabisulfite sodium, methionine,monothioglycerol (thioglycerol), nitrogen, propyl gallate, sulfitesodium, tocopherol alpha, alpha tocopherol hydrogen succinate,thioglycolate sodium, thiourea, anhydrous stannous chloride Solvents andBenzyl benzoate, oils, castor oil, cottonseed oil, N,Ndimethylacetamide, Co-Solvents ethanol, dehydrated ethanol,glycerin/glycerol, N-methyl-2-pyrrolidone, peanut oil, PEG, PEG 300, PEG400, PEG 600, PEG 3350, PEG 4000, poppyseed oil, propylene glycol,safflower oil, sesame oil, soybean oil, vegetable oil, oleic acid,polyoxyethylene castor, sodium acetate- anhydrous, sodium carbonate-anhydrous, triethanolamine, deoxycholate Buffers and Acetate, ammoniumsulfate, ammonium hydroxide, arginine, aspartic acid, pH-Adjusting benzesulfonic acid, benzoate sodium/acid, bicarbonate-sodium, boric Agentsacid/sodium, carbonate/sodium, carbon dioxide, citrate, diethanolamine,glucono delta lactone, glycine/glycine HCl, histidine/histidine HCl,hydrochloric acid, hydrobromic acid, lysine (L), maleic acid, meglumine,methanesulfonic acid, monoethanolamine, phosphate (acid, monobasicpotassium, dibasic potassium, monobasic sodium, dibasic sodium andtribasic sodium), sodium hydroxide, succinate sodium/disodium, sulfuricacid, tartarate sodium/acid, tromethamine (Tris) Stabilizer Aminoethylsulfonic acid, asepsis sodium bicarbonate, L-cysteine, dietholamine,diethylenetriaminepentacetic acid, ferric chloride, albumin, hydrolyzedgelatin, insitol, D,L-methionine Surfactant Polyoxyethylene sorbitanmonooleate (TWEEN ® 80), Sorbitan monooleate, polyoxyethylene sorbitanmonolaurate (TWEEN ® 20), lecithin, polyoxyethylene-polyoxypropylenecopolymers (PLURONICS ®), polyoxyethylene monolaurate,phosphatidylcholines, glyceryl fatty acid esters, urea Complexing/Cyclodextrins (e.g., hydroxypropyl-B-cyclodextrin, sulfobutylether-B-Dispersing cyclodextrin) Agents Viscosity Sodium carboxymethylcellulose, acacia, gelatin, methyl cellulose, Building Agents polyvinylpyrrolidone

Exemplary properties of various additives are summarized in Table 5below.

TABLE 5 Properties of Exemplary Additives Osmo/ Cryo Surfac- Cell Misci-Protec- tant/ Perme- Agent Lipolytic ble tant Solvent able Glycerol X XX X Urea X X X Ethylene Glycol X X X X PEG X X TRITON ™ X-100 X XDetergent Propylene Glycol X X Ethanol X X Polyvinyl Alcohol X X XAdonitol X X Erythritol X X Dextran (Glucan) X X Dextrose 20% in X X XWater (D20W) (Free Water Solution) Mannitol (Sugar X Alcohols) Sucrose(Sugar) X X X Hetastarch (Colloid) X X Epinephrine X X (Vasoconstrictor)PLURONIC ® X X (Poloxamers) SPAN ® (Sorbitan X X Esters) TWEEN ® X X(Polysorbates) CREMOPHOR ® X X (Polyethoxylated Castor Oil) Caffeine X XCholate X X X Deoxycholate X X X Lecithin X X X X Yohimbine X XGenistein X (Pro- X Apoptotic Agent) Resveratrol X (Pro- X ApoptoticAgent) Amino/ X X Theophylline Amino Acid X X (e.g.; Betaine) PolyvinylPyridine X X (PVP) Emulsions of Natural X Oils Intralipid X ElectrolytesX (Crystalloids) Calcitrol (Potential Cryosensitizer)

As used herein, “intralipid” refers to an emulsion of lipids typicallyused for intravenous nutrition, e.g., an emulsion of 20% intravenous fatemulsion, 20% soybean oil, 1.2% egg yolk phospholipids (lecithin), 2.25%glycerin, water, and sodium hydroxide to adjust pH. A variety of otherintralipid formulations are used in medicine.

Other additives include sugars, monosaccharides, disaccharides,oligosaccharides, polysaccharides, carbohydrates, lipids,anti-metabolites, oils, natural oils (e.g., canola, coconut, corn,cottonseed, flaxseed, olive, palm, peanut, safflower, soybean, and/orsunflower oil), peritoneal dialysis solution, ions (e.g., calcium,potassium, hydrogen, chloride, magnesium, sodium, lactate, phosphate,zinc, sulfur, nitrate, ammonium, carbonate, hydroxide, iron, barium, andthe like), and the like.

Foams and Foamy Slurries

Referring now to FIGS. 26A and 26B, slurry with 5% TWEEN® 20(polysorbate 20) in lactated Ringer's solution plus 5% dextroseinitially had a regular slurry consistency, but became very foamy whenreblended. Without being bound by theory, it is believed that foamyslurries can be formed with other detergents.

Foamy slurries or other biocompatible foams can be utilized asinsulators to further protect adjacent tissue from cold-induced damage.For example, a foamy slurry or other biocompatible foam can first beinjected adjacent to a cooling target (e.g., a nerve). A slurry having ahigher cooling power can then be injected adjacent to the target andwithin the foamy slurry or biocompatible foam as depicted in FIG. 27.The foamy slurry or biocompatible foam will act as an insulator, in partdue to the entrained air, thereby protecting adjacent tissue fromcold-induced damage and shielding the second slurry from warming byadjacent tissue.

Methods of Preparing Slurries

Slurries can be prepared using a variety of methods.

In one embodiment, a slurry is prepared using a commercially-availableice slurry generator such as those available under the MODUPAK™DEEPCHILL™ trademark from Sunwell Technologies Inc. of Woodbridge,Ontario. Commercially-available slurry generators include scrapedsurface generators that wipe away (e.g., with blades, augers, brushes)small ice crystals from a chilled surface and mix with water, directcontact generators in which an immiscible primary refrigerant evaporatesto supersaturate the water and form small smooth crystals, and supercooling generators in which water is supercooled and released through anozzle into a storage tank.

FIG. 1 depicts one exemplary method 100 of preparing a slurry.

In step S102, ice is obtained. The ice is preferably sterile ice and caneither consist purely or essentially of water or can be a frozen mixtureof water and one or more additives as discussed herein.

In step S104, one or more additives are optionally combined with the iceprior to processing. The one or more additives are preferably at or nearthe desired slurry temperature in order to prevent melting andrefreezing of the ice particles.

In step S106, the ice is processed into smaller pieces. A variety oftechniques and devices can be utilized to reduce the ice size includinga blade grinder (e.g., a blender, a food processor, and the like) thathave rotating blades, an ice crusher, an ice shaver, a mill, or othersuitable device. Suitable blade grinders are available under the WARING®trademark from Conair Corporation of Stamford, Conn. Suitable icecrushers and ice shavers are available under the CLAWSON™ trademark fromthe Clawson Machine Division of Technology General Corp. of Franklin,N.J. and under the SEMCO™ trademark from Semco Inc. of Pharr, Tex.Suitable mills include the benchtop analytical mill available fromCole-Parmer of Vernon Hills, Ill. and depicted in FIG. 18 and can beutilized to mill either ice or dry ice. In still another embodiment, iceparticles can be formed by grinding/milling ice between surfaces (e.g.,discs or screens) rotating in opposite directions. In yet anotherembodiment, shock waves, vibration (e.g., ultrasonic vibration), and/orthermal shock (e.g., from lasers or steam jets) can be used to fracturethe ice. In still other embodiments, the ice (and any additives) can beplaced in a bag and struck repeatedly with a mallet or other implement.

In step S108, one or more additives (e.g., glycerol) are added tocrushed ice (and any previously added additives).

In one example of this method, ice can be scraped at −80° C. into abiocompatible liquid cooled to 1° C. above the biocompatible liquid'sfreezing point. A surfactant cooled to 20° C. is then added and theresulting slurry is stirred vigorously.

FIG. 2 depicts another exemplary method 200 of preparing a slurry.

In step S202, sterile water is obtained. Sterile water is available froma variety of sources including Hospira, Inc. of Lake Forest, Ill.

In step S204, one or more additives are optionally combined with thesterile water prior to processing.

In step S206, the water (and any additives) are frozen. A variety oftechniques and devices can be utilized to freeze the water.

One example is an ice cream maker that utilizes a moving element (eithera paddle or a rotating vessel) to generate small ice crystals. Anexperimental prototype using a household ice cream maker is depicted inFIG. 3.

In another embodiment, small droplets of water (and optionallyadditives) are formed and then frozen. Suitable devices for formingsmall droplets of water include atomizers, injectors, ejectors,aspirators, Venturi pumps, nebulizers, humidifiers, ultrasonichumidifiers, and the like. The generated water droplets can beintroduced into a cold environment that can be achieved, for example,using dry ice. An example of ice produced by introducing dropletsgenerated by an ultrasonic humidifier into a dry ice environment isdepicted in FIG. 4.

In still another embodiment, small droplets of water (and optionallyadditives) are dropped into liquid nitrogen then harvested. Ice balls502 harvested using this technique are depicted in FIG. 5. This processcan be automated through the use of a microdropper such as thoseavailable from microdrop Technologies GmbH of Norderstedt, Germany.

In still another embodiment, the slurry components can be providedpre-mixed liquid in a bag (e.g., an intravenous fluid bag and like) andthen frozen within the bag under continuous or intermittent agitation,e.g., by shock waves, vibration (e.g., ultrasonic vibration), thermalshock (e.g., from lasers, steam jets), and the like to produce a slurrywithin the bag that can then be injected. This approach advantageouslyprovides a “closed” system for creation of slurries to promotesterility.

In still another embodiment, a plurality of ice particles can be formedin a sub-millimeter (e.g., having a largest cross-sectional dimension ofabout 0.1 mm or less) or micro-scale casting mold as depicted in FIG.31. The casting mold can be fabricated through molding, negativemolding, 3D printing, additive manufacturing, machining, and the like todefine receptacles of a variety of shapes and can be fabricated from avariety of materials such as polymers, plastics, elastomers, silicone,silicon, metals, and the like. The tray can be provided pre-loaded withice particles or can be loaded with water and frozen in a lab. The traycan flex to release the ice particles into a liquid component to form aslurry.

The casting can be performed by rapid cooling of the casting mold whilein contact with liquid water or flowing of liquid water over or throughthe cold casting material, during which ice forms within casts. Iceparticles can be removed from the casts by deformation of the castingmaterial using mechanical strain, stress waves, or shock waves. Iceparticles can be removed from the casts by partial melting from anexternal or internal energy source. Ice particles can be removed oraided to be removed from the casts by centripetal force, e.g., bycentrifugation. For example, the casting mold can be rapidly rotatedwhile cooled and periodically supplied with water in order to createsmall ice particles near the mold surface that are thrown off bycentripetal force into a cold environment for collection.

Slurry Storage and Further Processing

Both the slurries described herein and precursor ice particles can bestable for years if held at stable humidity and temperature below thefreezing point of the solution or the ice particles. In order to guardagainst growth or agglomeration of ice crystals, it is preferable tostore the slurry at a stable temperature below the temperature forintended use and allow for partial melting of the slurry to reach thedesired injection temperature prior to use. Partial melting can beachieved by warming of the slurry (e.g., by exposure of a vesselcontaining the slurry to ambient conditions or by actively applying anenergy source) or introducing additional solute (e.g., glycerin).

Various techniques can be employed to prevent or minimize sublimation ofeither precursor ice particles or slurries during storage, transport,and/or handling. For example, ice particles can be coated with asurfactant that acts as a barrier to sublimation and reduces frictionbetween ice particles. Additionally or alternatively, the ice particlesand/or slurries can be stored under elevated pressure (e.g., abovewater's triple point).

Either method described above can be performed by a single actor at asingle location at a single time or can be performed by one or moreactors at one or more locations at one or more times. For example, smallstable ice particles can be packaged and shipped using standard coldshipping methods and stored in a standard freezer (e.g., at −20° C.).The ice particles can be combined with one or more additional additivesin the clinic shortly or immediately prior to injection. As discussedherein in greater detail, the additives can, for example, bebiocompatible solutions, contain a biocompatible surfactant such asglycerol, and be precooled (e.g., to a temperature approximating thedesired temperature of the slurry at the time of injection).

The additives can be added through a variety of methods. In oneembodiment, the ice particles are stored in a container such as aplastic bag such as those commonly used to store intravenous (IV) fluidand the additive is injected, pumped, or allowed to flow by gravity intothe ice particles. In other embodiments, the additive is poured over theice particles. In still another embodiment, the additive is provided infrangible or burst pouch within the same container as the ice particles.This frangible or burst pouch can be squeezed to rupture the pouch andcombine the additive and the ice particles at the desired time.

Once the ice particles and the additives are mixed de novo at the pointof care or pre-mixed slurries are removed from a freezer, thetemperature is preferably monitored to either maintain or achieve adesired temperature. For example, the slurry may need to rise to adesired temperature, but it may be preferred that the slurry does notrise significantly beyond a desired temperature. Various thermometers,thermocouples, and other temperature measuring devices can be used tomeasure the temperature of the slurry. These measurements can beinternal or external to the container holding the slurry. In oneembodiment, a liquid crystal thermometer is applied to the outside ofthe container (e.g., an IV bag) holding the slurry. Suitable liquidcrystal thermometers capable of measuring temperatures between −30° C.to 0° C. are available under Part No. 427-1 from Telatemp Corporation ofAnaheim, Calif. In another embodiment, an additive in either the slurryor the container holding the slurry can change color to indicate anappropriate and/or inappropriate temperature. Similarly, a temperaturemonitor can be used to indicate inappropriate storage or transportconditions for the slurry.

Slurry Delivery

The injectable slurry can be introduced using various parenteraldelivery systems and techniques including gravity flow, injectionthrough a syringe, a cannula, a catheter, tubing, and/or a pump, and thelike. A control device can control the flow rate, volume, and orpressure of the injected slurry in order to extract a desired amount ofheat from tissue adjacent to the injection site.

Optionally, an imaging technique such as ultrasound, magnetic resonance,x-ray, and the like can be utilized to verify the proper positioning ofthe injection device and/or the slurry. In particular, ice is a verystrong reflector of ultrasound, while lipid rich cells are poorreflectors of ultrasound. Ultrasound imaging is a convenient “bedside”imaging modality with sufficient contrast and depth of imaging to guideand/or monitor administration of the slurry.

Therapeutic Applications of Injectable Slurries

The injectable slurries described herein can be utilized to target alltissue types including, but not limited to, connective, epithelial,neural, joint, cardiac, adipose, hepatic, renal, vascular, cutaneous,and muscle tissues. The injectable slurry advantageously can focus acooling effect direct at the site of the targeted tissue without thechallenges of diffusion of heat or perfusion tissue.

Referring now to FIG. 6, a general method of treatment 600 usinginjectable slurries is provided. Although depicted in a linear manner,step(s) can be omitted, repeated, or executed in various orders.

In step S602, the amount of heat to be extracted is determined. Theamount of heat to be extracted can be routine and predictable,particularly for treatment of small structures such as nerves and can bespecified a priori, e.g., as part of approval of a medical device orprocedure, as part of the instruction manual for a medical device,and/or as a preset control parameter in a medical device. In otherembodiments, the amount of heat to be extracted can depend on the amountof tissue to be treated, the location of the treatment site, and otherattributes of the subject.

In step S604, the treatment parameters are selected. Treatmentparameters can include the composition of the slurry (e.g., the icecontent and additive content) and the temperature, which together(particularly the ice content) determine the cooling power of theslurry. (The additive content largely influences the temperature of theslurry.) Exemplary ice content and additive content is discussed herein.Exemplary temperatures of the slurry include about +10° C., about +9°C., about +8° C., about +7° C., about +6° C., about +5° C., about +4°C., about +3° C., about +2° C., about +1° C., about 0° C., about −1° C.,about 2° C., about −3° C., about −4° C., about 5° C., about −6° C.,about 7° C., about 8° C., about 9° C., about 10° C., about 11° C., about12° C., about 13° C., about 14° C., about 15° C., between about 15° C.and about 25° C., between about 25° C. and about 50° C., between about50° C. and about 75° C., and the like.

Without being bound by theory, Applicant notes that heat of fusion forlipids is about half of the heat of fusion for water and believes thatabout 2 units of fat volume can be treated with about 1 unit of slurryvolume. It may be desirable to be conservative in the amount of slurryinjected, particularly when the treatment site is proximate to nerves,blood vessels, organs, and the like.

In step S606, the slurry is optionally allowed to reach the desiredtemperature. This can be achieved by allowing the slurry to sit at roomtemperature or in a controlled temperature environment until itstemperature rises to the desired temperature. The slurry can beoptionally be stirred or agitated to promote an even temperaturedistribution. The slurry can optionally be placed in an insulatedcontainer to preserve the slurry at the desired temperature. Likewise,the catheters, needles, and/or tubing used to deliver the slurry to thesubject can optionally be insulated (e.g., with dead-air space orrubbers such as neoprene) to minimize temperature rise.

In step S608, vasoconstriction is optionally applied, e.g., throughphysical means such as suction or pressure or chemical means such asepinephrine injections.

In step S610, the application site is optionally pre-cooled to minimizemelting of the slurry upon injection. In one embodiment, one or morethermoelectric (Peltier) cooling devices can be used to pre-cool tissueto a desired temperature prior to injection of a slurry. Suitablethermoelectric coolers are available from TE Technology, Inc. ofTraverse City, Mich. and Quanta Aesthetic Lasers USA of Englewood, Colo.

In step S612, one or more cryoprotectant approaches can optionally beemployed.

For example, energy can be applied to tissue adjacent to the targettissue in order to protect the adjacent tissue from undesired coolingand/or enable more aggressive cooling of the target tissue. Energy canbe applied simultaneous with the slurry, in an oscillating manner, inresponse to feedback, and the like. Suitable energy sources includeradiofrequency (RF) energy generating units that can be adapted,configured, and/or programmed to generate monopolar, bipolar,capacitively-coupled, and/or conductively-coupled RF energy. The RFenergy can have a frequency between about 0.3 MHz and about 100 MHz.Other suitable energy sources include coherent light sources, incoherentlight sources, heated fluid sources, resistive (Ohmic) heaters,microwave generators (e.g., producing frequencies between about 915 MHzand about 2.45 GHz), and ultrasound generators (e.g., producingfrequencies between about 300 KHZ and about 3 GHz).

In another example, one or more cryoprotectants (e.g., glycerol,propylene glycol, and the like) can be applied to protect non-lipid-richtissue from cold-induced injury. The cryoprotectant can be appliedtopically to the epidermis and/or can be injected into a desired region.

In step S614, an injection device is inserted into the subject. Asdiscussed herein, suitable injection devices include hypodermic needles,cannulas, catheters, and the like. The location and depth of insertioncan vary to reflect the target region. For example, the injection devicecan be inserted to an appropriate depth for parenteral, subcutaneous,intramuscular, or interstitial injections.

In step S616, the location of the injection can be verified, e.g.,through ultrasonic or x-ray imaging. Alternatively, the slurry can beadministered through a “blind” injection.

In step S618, a foamy slurry or biocompatible foam is optionallyinjected as described herein to insulate a later injected slurry.

In step S620, feedback about the injection is obtained. Feedback caninclude additional imaging of the slurry, resistance to furtherinjection, input from the patient (particularly when the slurry containsan anesthetic), information about the treatment site (e.g., using thefeedback devices described in U.S. Pat. Nos. 7,367,341 and 8,840,608)and the like. For example, ice particles in the slurry are radiopaqueand can be readily visualized using ultrasound.

In step S622, the slurry is injected. Applicant has found that handpressure typical of that provided to a syringe is sufficient forinjection, but higher mechanical pressure can be used as well. Theslurry can be injected in discrete, pre-determined volumes or can beinjected until a medical professional determines that the injectionshould cease, e.g., due to increased pressure or resistance to furtherinjection or having injected a desired volume of slurry.

In step S624, feedback about the injection is obtained.

In step S626, the slurry is optionally withdrawn, for example using themethods and devices described in U.S. Patent Application Publication No.2013/0190744. In many cases, particularly subdermal injections, theslurry will be left in the body to melt and be absorbed. In someembodiments, the suction can be applied concurrently, intermittently,and/or alternatingly to injection of a slurry. For example, a filteredor smaller gauge cannula can be inserted adjacent to or coaxial with aninjection device as depicted in FIGS. 30A-30C to remove melted fluidduring a procedure while leaving most or all of the ice particles in theinjection site. Such an embodiment can be particularly useful foranatomically constrained targets such as nerves in which injecting largevolumes of slurry poses challenges.

In step S628, the injection device is removed from the injection site.

In step S630, the injection site is assessed. Assessment can beperformed during or after the procedure to assess the effect of theinjected slurry. Assessment can be performed by a medical professionaland/or by the subject and can include self-reporting, photography,calipers, MRI, as well as the imaging devices described in U.S. Pat.Nos. 7,367,341 and 8,840,608.

All or portions of this method can, but need not, be repeated one ormore times for the same injection site and/or target tissue. Multipleinjections can be performed in a serial, overlapping, or parallelmanner.

In one embodiment, the cryoslurry is injected at a sufficient volume tocause tumescent swelling of the injection site. In another embodiment, aplurality of injection sites each receive an effective amount of slurryfor treating a small region of tissue. For example, injections ofsubstantially uniform volumes of slurry can be made in a grid or otherpattern.

In some embodiments, serial injections are made within a singletreatment session. For example, a first injection can be made topre-cool the area before a second injection is made to achieve thedesired clinical or cosmetic cooling effect. The formulation of theseserial injections can vary. For example, the second injection can have ahigher or lower cooling power than the first injection.

Cryoneurolysis

A major limitation of medical nerve blocks is their limited duration,which last for hours but not even one day. When somewhat longer reliefof pain is needed, constant or repeated infusions of anesthetics aresometimes performed through a needle or through an indwelling catheter,but typically chronic pain is the setting for use of potent analgesicssuch as opiates, with associated high risk of side effects includingaddiction.

As discussed in U.S. Provisional Patent Application Ser. No. 62/042,979,filed Aug. 28, 2014, cold and particularly slurries can be used toprovide long-term, but reversible inhibition of nerve function. Theslurries described herein can provide unprecedented long-lastingreduction or full relief of pain, regardless of its cause.

Methods of the invention can also be used to reduce or eliminatesymptoms associated with pain disorders caused by surgery, such as anysurgery that makes an incision through the skin and induces pain. Thisincludes thoracic surgery pain (e.g., treatment of incisional surgicalpain) caused by thoracic surgery. The slurry can be injected prior,during or after incision.

Methods of the invention can also reduce or eliminate symptomsassociated with motor disorders including, but not limited to,hemifacial spasm, laryngospasm and gustatory hyperhidrosis. Methods ofthe invention can also be used to reduce muscle spasms caused byaberrant nerve firing such as bladder or facial spasms. Methods of theinvention can also be used to target motor nerves if prolonged paralysisof a motor nerve is desired.

The solution comprising the slurry can be administered to the peripheralnerves of the subject by injection, infusion or tumescent pumping of theslurry into a nerve or nerves such as peripheral, subcutaneous orautonomic nerves of the subject by injection into a nerve or nervesselected from the group consisting of the cutaneous nerve, trigeminalnerve, ilioinguinal nerve, intercostal nerve, interscalene nerve,supraclavicular nerve, infraclavicular nerve, axillary nerve, pudendalnerve, paravertebral nerve, transverse abdominis nerve, lumbar plexusnerve, femoral nerve, and sciatic nerve.

Methods of the invention can also reduce or eliminate pain associatedwith a nerve plexus (i.e., a group of intersecting nerves) including butnot limited to the cervical plexus that serves the head, neck andshoulders, the brachial plexus that serves the chest, shoulders, armsand hands, the lumbar plexus that serves the back, abdomen, groin,thighs, knees, and calves, the sacral plexus that serves the pelvis,buttocks, genitals, thighs, calves, and feet, the celiac plexus (solarplexus) that serves internal organs, the coccygeal plexus that serves asmall region over the coccyx, the Auerbach's plexus that serves thegastrointestinal tract and Meissner's plexus (submucosal plexus) thatserves the gastrointestinal tract.

Methods of the invention can also be used for renal sympatheticdenervation, which is an emerging therapy for the treatment of severeand/or resistant hypertension.

Injecting a physiological slurry around a particular sensory nervespecifically blocks conduction of that nerve, leading to sensory lossesin the entire distribution of the target nerve. Thus, slurry injectioncan relieve pain and/or itch over a very large region of skin, muscle,joints, and the like that are “served” by a particular target nerve. Inprevious animal studies, it has been shown that cooling can also blockmotor nerves. Many nerves in the human body have mixed sensory and motorfunction. For a mixed nerve, it is often desirable to block sensationbut not motor function. (Temporary motor loss may be less of a concernif the subject has already lost motor function, e.g., throughamputation.) The anatomy of the peripheral nervous system is such thatthe motor and sensory nerve fibers become fully separated near thespine. A prolonged and specific block of sensory functions associatedwith spinal nerve level(s), can be obtained by prolonged block of thesensory nerve root using slurry injection. Slurry injection, with orwithout ultrasound guidance, to the sensory portion of the peripheralnerve can provide prolonged, specific relief of pain. The step ofinjection for slurry is similar to injection of medical anesthetic nerveblocks.

It is further noted that the slurries described herein can be utilizedto provide long-term, but reversible inhibition of the autonomicsympathetic nerves adjacent to the renal artery to treat hypertension.Other exemplary nerve targets include the sympathetic andparasympathetic nerves.

Cryolipolysis

The slurries described herein can be utilized to provide selectivereduction of lipid-rich cells.

Slurry injection provides several improvements over cryolipolysis usingexternal cooling device for subcutaneous fat reduction, due, in part, tothe high cooling power of injectable slurries. First, local slurryinjection requires only a few minutes of active time to achieve thedesired temperatures. Second, slurry injection appears induce the lossof a greater thickness of subcutaneous fat more rapidly (e.g., within 3weeks to 5 weeks). The greater efficacy probably arises because, unlikesurface cooling, slurry extracts heat directly from the target tissuelayer, and provides a much faster cooling rate in the target tissue.Third, slurry injections can avoid the epidermis and dermis andapproaches that are commercially utilized to detect epidermaltemperatures and protect against damage to non-lipid-rich cells. Forexample and without being bound by theory, it is believed that thedermis and/or epidermis will remain within 15° C. of normalphysiological temperature during most subcutaneous slurry injectionsinto adipose tissue. Before, during, and/or after slurry injection intosubcutaneous tissue, warming of the skin can be performed, e.g., byapplication of a warm object or by radiant heating. Fourth, slurryinjection is not limited for depth or location of treatment. Slurry canbe injected superficially, deeply, or throughout the subcutaneous fatlayer and is not limited by the geometries of the subject's skin and thetopical applicator. Fifth, slurry injection can be done with greateranatomic accuracy. Physicians are generally very skilled at performinginjections, even without ultrasound guidance. With ultrasound guidance,in which the needle cannula or catheter used for injection can bedirectly seen, slurry can be placed with high accuracy. As noted above,the slurry itself can be visualized by ultrasound, such that the treatedtissue is well-defined and known during treatment. Finally, slurries canbe administered without the use of large thermoelectric devices used incommercial cryolipolysis systems.

Slurry injections can also quickly numb the nerves adjacent to thetreatment site(s), potentially eliminating or reducing the use ofanesthesia used in commercial cryolipolysis procedures.

Without being bound by theory, it is believed that cryolipolysis can beachieved through injections of slurries at temperatures of about +5° C.and lower. Without being bound by theory, it is believed that higher icecontent slurries will be most effective in extracting sufficient heat toachieve cryolipolysis in clinically and/or cosmetically significantnumbers of adipose cells. For example, slurries having at least about50% (±30%) ice by weight may be preferred. In order to improve theinjectability of slurry and the ability of slurry to infiltratesubcutaneous adipose tissue, chemical (e.g., freezing point depressants)and thermal (e.g., warmer coolant) techniques can be used to make anydendritic shaped ice particles more globular. Prior to injection ofslurry, addition of a biocompatible exothermic solute may be added tofurther decrease the temperature of slurry. In some embodiments, theslurry includes an effective amount of a lipolytic agent such as thosedescribed herein.

The amount of slurry and/or the slurry ice content to be injected can becalculated and calibrated to produce a desired amount of cryolipolysis.Without being bound by theory, it is believed that cooling effects ofslurries can be tightly controlled such that two slurries having thesame composition and physical characteristics will produce substantiallythe same amount of cryolipolysis if injected into the same locationunder the same physiological conditions.

Without being bound by theory, it is believed that the slurriesdescribed herein can be utilized to achieve cooling rates above about 2°C. per minute. For example, the cooling rate can be greater than about10° C. per minute, greater than about 20° C. per minute, greater thanabout 30° C. per minute, greater than about 40° C. per minute, greaterthan about 50° C. per minute, greater than about 60° C. per minute,greater than about 70° C. per minute, greater than about 80° C. perminute, greater than about 90° C. per minute, greater than about 100° C.per minute, greater than about 110° C. per minute, greater than about120° C. per minute, and the like. For example, Applicant has achievedcooling rates of about −117° C. per minute in tissue in contact withslurry and about and −26° C. per minute in adjacent adipose tissue.Cryolipolysis via slurry injection can be applied to variety of regionsin which fat reduction is desired for medical and/or cosmetic reasons.Exemplary regions include the abdomen, flanks (also known as “lovehandles”), buttocks, thighs, arms, neck, chin (e.g., treatment ofsubmental fullness also known as a “double chin”), and the like.

Treatment of Obstructive Sleep Apnea

Sleep apnea is due to upper airway obstruction during sleep. Sleep apneacauses poor quality sleep, wakening, organ damage from hypoxia(including myocardial infarctions, stroke, and cumulative brain injury),and is a frequent cause of death in obese individuals. The prevalence ofsleep apnea has been steadily increasing in the US due to obesity.Present treatments are generally aimed at reducing the degree of obesity(through diet, exercise, medications, and/or surgery) and at keeping theairway open during sleep. Continuous positive airway pressure (CPAP)helps to keep the airway patent, but requires wearing a close-fittingmask and pressure apparatus all night. These often fall off duringsleep, leak, or are uncomfortable enough that sleep can be disruptedsimply by wearing them. Occlusion of the airway is strongly related tothe amount of fat located in deep fat pads located at the base of thetongue and along the soft palate and lateral pharynx. Surgicalprocedures have been developed, for example, to suspend the palate ordebride pharyngeal fat, but these ultimately also cause scarring, oftenfail to open the airway sufficiently, are painful and cause local edemaduring healing that can precipitate worse sleep apnea, airwayobstruction, respiratory distress and death.

Injection of physiological ice slurry into the subglottal, palataland/or pharyngeal fat is a novel treatment for sleep apnea. Guidance byultrasound for accurate placement and injection of slurry within thetarget tissue is also a novel method. These fat compartments aredistinct from subcutaneous fat. They can be visualized by ultrasound, asregions of echolucency (low signal) compared with surrounding muscle,fascia and other structures. Slurry with a high ice content isparticularly desirable to minimize the volume of injected slurry neededfor effective reduction of the target fat. During about 6 weeks aftertreatment, injection of slurry into the fat will induce gradualreduction in the amount of fat, resulting in improvement or cure of thepatient's sleep apnea. The intrinsic selectivity of this treatment forfat is such that adjacent muscle, fascia, salivary gland and othertissues are spared from injury, while reliable reduction of the targetfat can be achieved. Unlike surgery, this would be an office procedureperformed with little or no anesthesia. Unlike surgery, there would beno scarring because the treatment is intrinsically selective for thelipid-rich adipose tissue causing sleep apnea. The post-treatment pain,inflammation and risk of airway compromise will be less than surgicalprocedures, because adjacent tissues are not affected. Unlike CPAP,injection of physiological ice slurry provides a permanent improvementand does not interfere with sleep.

Exemplary regions that can be targeted include the anterolateral upperairway, pharyngeal fat pads (for example, fatty deposits in thelaryngopharynx, nasopharynx, oropharynx, and palatopharynx),parapharyngeal fat pads (for example, fatty deposits in the retropalataland retroglossal regions), fat located within the tongue (e.g., withinthe posterior tongue), and soft palate. Without being bound by theory,it is also believed that treatment with the slurries described hereinwill thicken septa and tighten skin, which can also reduce the tendencyof the airway to collapse.

Injections can be made through the mouth or through the neck in order tobest target a particular region of fat while avoiding adjacent nerves,blood vessels, and other structures. The amount of fat removed pertreatment can be adjusted by adjusting the volume and/or ice content ofthe injected slurry, and precise location of the fat removed can beadjusted by location of the injection(s). Multiple courses of treatmentthat each remove a small volume of fat from fat located in the tongue,neck, palate, pharynx and/or tonsil may be preferred in order tominimize temporary airway constriction due to the added slurry volume.

This novel method for treatment of sleep apnea can have a major impacton health care, including reducing the morbidity, heart attacks, strokeand death associated with obesity.

Treatment of Spinal Cord Lipomas and Lipomyelomeningocele

Spinal cord lipomas and lipomyelomeningocele are both associated withabnormal fat accumulation in and around the spinal cord. A spinal cordlipoma is fat within the normally positioned spinal cord without anyskin or bony abnormalities. These lesions are most commonly locatedwithin the thoracic spinal cord. Although rare, these lesions can causesevere morbidity. They may be symptomatic and appear most often inadults. Patients can present with spinal cord compression that can causenumbness and tingling, weakness, difficulty with urinating or bowelmovements, incontinence, and stiffness of the extremities.

The current treatment for symptomatic lipomas around the spinal cord thetreatment of choice is a laminectomy to gain access to the spinal cord.The goal of surgery is to reduce the size of the lipoma, not totalremoval of the fat. No other treatment method is recommended.

Embodiments of the invention utilize injection of cold slurry tospecifically target the spinal lipomas without the need to dolaminectomy as cold slurry could be directly injected into the lipomathrough a needle. With the use of ultrasound guidance, the lipoma can belocated and specifically destroyed via a cold slurry injection. Thisnovel method of treating lipomas will reduce the morbidity associatedwith surgical procedures

The slurry injection method of treatment can be used for any lipomasaround the nerves including peripheral nerves. Lipomas may also grownear or surrounding important peripheral nerves. Therefore, theirremoval can cause nerve injury and possible paralysis. Common locationsinclude the neck, buttock, and forearm. Again, injection of the slurryinto the lipomas to selectively target them will reduce the need forsurgery.

Lipomyelomeningocele (LMM) is a common and severe closed neural tubedefect in children. Lipomyelomeningocele lies within the spectrum ofclosed neural tube defects. It represents a complex disorder that maypresent with neurological deficits secondary to the inherent tetheredcord. This is a lesion present at birth that is commonly associated withspina bifida (congenital failure of the spinal bones to close). Thecondition is associated with abnormal fat accumulation that starts belowthe skin and extends through the bony opening to the spinal cord. Theselesions become evident within the first few months to years of life andaffect females more than males in a 1.5 to 1 ratio.

More than 90 percent of patients will have an obvious soft tissueswelling over the spine in the lower back. These lesions are covered byskin and are not painful. Patients may lose neurological function withinthe first few weeks after birth, but more typically, functiondeteriorates over a period of months to years. Neurological symptomsusually include weakness and bladder and bowel incontinence. Theweakness can be symmetrical or asymmetrical and can result in atrophy ofthe lower extremities. In older adolescents and adults, pain may be thedriving force to bring the patient to a doctor. The pain may radiate andbe difficult to describe. Back mobility may be limited.

Surgery is the treatment of choice whenever possible, but most cases areinoperable. The goals of surgery are to release the attachment of thefat (tethering) to the spinal cord and reduce the bulk of the fattytumor. With surgery, 19 percent of patients will improve, 75 percentwill be unchanged, and 6 percent will worsen. A fat-selective, minimallyinvasive treatment is likely to produce safety and efficacy that aresuperior to surgery, and would make it possible to treat inoperablecases.

Injection of cold slurry can specifically target the lipoma and decreaseits size, thus preventing neurological damage associated with theirgrowth. The injection needle can be guided by ultrasound or MRI(magnetic resonance imaging) for accurate placement and injection ofslurry within the target tissue. MRI can provide an accuratepre-treatment “map” of the local anatomy including bones, spinal cordand the target lipomeningiocele. The use of slurry to treat LMM willoffer a novel, less morbid and lifesaving treatment for these pediatricpatients.

Breast Reduction

Pseudogynecomastia or lipomastia in males is due to the presence of fatdeposits in the breast. This condition is more prevalent in men withaging, overweight, certain drugs, or exposure to estrogens includingdietary sources. Currently, surgical removal is the preferred treatment.Applicant believes that injection of embodiments of the slurriesdescribed herein into the excess fat around the breast tissue will beless invasive technique with less morbidity.

Additionally, slurry injections can be used for female breast reductionprocedures, particularly, as a substitute to liposuction-only techniquesindicated for minor-to-moderate volume reduction. The increase inconnective tissue discussed in greater detail herein also can provide afirming and lifting effect to either the breast or pectoral region.

Treatment of Epicardial and Pericardial Fat

The slurries described herein can be utilized to treat epicardial and/orpericardial fat. Such treatments can be used for the prevention ofcoronary artery disease and coronary atherosclerosis, prevention andtreatment of atrial fibrillation and atrial tachyarrhythmias, andprevention and treatment of ventricular tachyarrhythmia.

Thoracic fat includes extra-pericardial (outside the visceralpericardium) and intra-pericardial (inside the visceral pericardium)adipose tissue. It is called ectopic adipose tissue although it is anormal anatomical structure. Intra-pericardial adipose tissue, which ispredominantly composed of epicardial and pericoronary adipose tissue,has a significant role in cardiovascular system function. The epicardialfat is located between the myocardium and visceral pericardium and thepericardial fat, is located outside the visceral pericardium and on theexternal surface of the parietal pericardium. Epicardial and pericardialfat are embryologically different.

Recent studies have suggested that increased epicardial fat could be animportant risk factor for cardiac disease. It secretes pro-inflammatorycytokines that can lead to development of coronary artery disease (CAD).In humans, there is a positive association between epicardial adiposetissue (EAT) volume and coronary atherosclerosis burden. Prospectivecase-cohort and case-control studies have shown that EAT volumepredicted future CAD events and myocardial ischemia. These findingsuggest that EAT might contribute locally to coronary atherogenesis. Inswine models, it has been shown that selective surgical excision ofadipose tissue in direct contiguity with one of the epicardial coronaryarteries attenuated the progression of atherosclerosis, thus suggestingthat removal of epicardial fat can serve as a preventive measure forCAD.

Pericardial fat may represent an important risk factor forcardiovascular disease because of its unique properties and itsproximity to cardiac structures. Pericardial fat has been associatedwith an adverse cardiovascular risk profile, coronary artery calcium,and prevalent cardiovascular disease in several studies. Pericardial fatvolume (PFV) has recently been reported to be strongly associated withCAD severity and presence. Pericardial fat has also been associated withcommon arrhythmias, such as atrial fibrillation (AF). AF is the mostcommon cardiac arrhythmia in clinical practice and is associated withmajor morbidity and mortality. AF prevalence has been projected toincrease in the coming decades and is expected to affect over 7.5million Americans by the year 2050. Pericardial fat has also beenassociated with ventricular tachyarrhythmia and mortality from systolicheart failure.

The slurries described herein can be utilized to treat epicardial and/orpericardial fat by injection of slurry into and/or around pericardialand/or epicardial fat during cardiac surgery. Additionally oralternatively, computed tomography (CT) or ultrasound (US) imaging canbe utilized to guide a needle into the pericardial and/or epicardial fatfor slurry injections. Injections can also be performed under directvision of video-assisted thorascopic surgery.

In still another embodiment, slurry can be injected into the pericardiumwith or without the use of ultrasound guidance. The main approaches toaccessing the pericardium are subcostal, parasternal and apical. In oneexample, pericardiocentesis can be performed using a long, thin needleor catheter (e.g., 7-9 cm, 18 G). At normal physiologic conditions,there is less than 50 ml of fluid in the pericardial space. In acuteconditions, this space can hold a volume of up to 200 ml withouthemodynamic compromise and can hold more than 500 ml if fluidaccumulates chronically. Hence, one could postulate that there is a safetherapeutic window in which volumes of slurry could be injected andeither removed from the pericardium or left in the pericardium.

Treatment of Visceral Fat

The slurries described herein can be utilized to provide selectivereduction of lipid rich cells such as visceral fat in accordance withthe methods described in U.S. Patent Application Publication No.2013/0190744. For example, the slurries described herein can beintroduced into the abdominal and/or peritoneal cavity. Such injectionscan reduce lipid-rich cells in structures such as the omentum and theperinephrium and regions such the perigonadal, retroperitoneal, andmesenteric regions of the body.

Non-Selective Cryoablation

In addition to selectively targeting lipid-rich cells in the methodsdiscussed above, embodiments of the slurries described herein can beutilized in traditional cryoablation techniques including prostatecryoablation, renal cryoablation, cardiac cryoablation, fibroadenomacryoablation, and the like. Traditional cryoablation is performed withvarious invasive probe devices at very low temperatures, typically about−30° C. to −100° C. Tissue destruction is not selective for lipid-richcells at these temperatures. An injected slurry with high osmolality andhigh ice content can achieve temperatures below −20° C., and could beused for these non-selective procedures, with some advantages overexisting cryogen probe devices.

Connective Tissue Enhancement

Selective loss of fat produces an increase in the relative amount ofconnective tissue at and/or near the site of slurry injection. Byaffecting primarily the adipocytes and removing fat, the connectivetissue septae that supported the fat remain and become thicker. This isclearly seen in both histology and gross tissue images from the swineexperiments. Fat is generally mobile and weakly supportive. The relativeincrease in connective tissue after treatment provides better supportfor the overlying skin. After cryolipolysis treatments, Applicantobserved clinically that laxity (sagging) improves dramatically.

In other words, adipose tissue is a connective tissue, but it tends tobecome lax. Slurry injection removes the adipocytes, but preserves andstimulates the septae, resulting in less laxity and greater mechanicalsupport. Histology and also gross images such as FIGS. 16A and 16Bclearly show that slurry injection into adipose tissue causes thischange.

This effect provides an added benefit to procedures such ascryolipolysis, breast reduction, and treatment of obstructive sleepapnea, pseudogynecomastia, and lipomastia, but can be used for the solepurpose of skin tightening.

Pelvic Floor Strengthening and/or Tightening

The pelvic floor is the supportive apparatus that holds the pelvicorgans in place. Pelvic floor dysfunction (e.g., due to pelvic floorlaxity) can cause abnormal defecation, urinary dysfunction, prolapse,pain, and sexual dysfunction.

The slurries and methods described herein can be applied to strengthenand/or tighten the pelvic floor. For example, slurries can be injected(e.g., through transurethral, transvaginally, or transperitonealinjections) adjacent to the pelvic floor to induce tightening and/orthickening of the pelvic floor in order to better support one or morepelvic organs.

Treatment of Urinary Incontinence

In a recent survey among women aged 25-84 in the United States, anestimated 15% report experiencing stress incontinence and 13% reportexperiencing urge incontinence/“overactive bladder.” These twoetiologies of incontinence are due to separate mechanisms, though bothmechanisms may be experienced by a single patient.

Stress incontinence is the most common type of incontinence in youngerwomen, often from urethral hypermobility, which is due to insufficientsupport from the pelvic floor. This lack of support is due to a loss ofconnective tissue. This loss of support is also associated with otherconditions such as pelvic organ prolapse and problems with defecation(both constipation and incontinence). Administration of the slurriesdescribed herein in the pelvic area can thicken the connective tissueand thereby increase the support of the pelvic floor. Thus, in oneembodiment, the invention provides a method for treating stress urinaryincontinence in a subject in need thereof. The method comprisesadministering to the connective tissue of the pelvic floor of thesubject a therapeutically effective amount of a slurry described herein.

In contrast, urgency incontinence is due to overactivity of the detrusormuscle. The slurries described herein can be used as an injectabletherapy to inhibit neural input to the bladder.

Strengthening of Abdominal Wall

Abdominal laxity can lead to hernias or cosmetically bothersomeabdominal protrusions.

The slurries described herein can be used to cause skin tighteningand/or tightening of the fascia supporting the abdominal wall to preventand/or remedy hernias or abdominal protrusions.

Perivascular and Periadventitial Adipose Tissue

As described herein, the slurries described herein can be utilized totreat cardiovascular diseases associated with epicardial and/orpericardial fat. In some applications, the slurries described herein maybe used to target perivascular or periadventitial adipose tissue withinthe human body for the purpose of modulation of adipose and immunefunction, as well as, targeted removal to provide treatment of variousdiseases or clinical states.

Almost all blood vessels are surrounded or embedded in perivascularadipose tissue (PVAT), which represents about 3% of the total bodyadipose tissue mass. PVAT was initially considered to play primarilymechanistic support for the vasculature but in recent years it hasbecome clear that PVAT plays a critical role for the regulation ofvascular/endothelial function in both physiology and pathology. In fact,most arteries prone to the atherosclerosis are all surrounded with PVAT.Anatomically, PVAT is contiguous with adventitial layer of the bloodvessel wall, and adipocytes migrating from the PVAT have been detectedwithin the adventitia. PVAT is also called periadventitial adiposetissue, and is shown to also play critical role in vascular remodelingand vascular disease. It is now believed that the crosstalk betweenPVAT, the adventitia, the endothelial and smooth muscle layers of bloodvessels are essential in normal vascular function and are perturbed indiseases such as arterial atherosclerosis, hypertension, arterialaneurysm formation such as aortic aneurysm, arrhythmia, arterial spasm,arterial ulcers. Circumstantial evidence also links PVAT to thepathogenesis of non-atherosclerotic vascular diseases includingneointimal formation, aneurysms, arterial stiffness andvasculitis/vasculitis syndromes, such as Takayasu's arteritis, whereinfiltration and migration of inflammatory cells from PVAT into thevascular wall may play a contributory role.

PVAT plays a critical role in the pathogenesis of cardiovascular andother vascular pathologies, in which it has been shown to becomedysfunctional with altered cellular composition and molecularcharacteristics. PVAT dysfunction is characterized by its inflammatorycharacter, oxidative stress, increased production of pro-inflammatoryparacrine factors such as resistin, leptin, IL-6, TNF-alpha, MCP-1,RANTES, and decreased production of vasoprotective factors such asadiponectin. The pro-inflammatory factors produced by dysfunctional PVATinitiate and promote inflammatory cell infiltration includingmacrophages, lymphocytes, dendritic cells, NK cells which propagate thevascular pathology. Local expansion of PVAT has been associated withatherosclerotic plaque formation, vascular calcification, hypertensionand aortic abdominal aneurysm.

PVAT inflammation in vascular disease are important in the context ofnumber of cardiovascular disorders including atherosclerosis,hypertension, vascular aneurysms, diabetes and obesity. Inflammatorychanges in PVAT's molecular and cellular responses are uniquelydifferent from visceral, subcutaneous and adventitial adipose tissue,highlighting the uniqueness of this adipose tissue compartment. Thedifferences between white, brown and perivascular adipose tissue arehighlighted in the publication by Nosalski R, Guzik T J. Perivascularadipose tissue inflammation in vascular disease. Br J Pharmacol. October2017; 174(20):3496-3513.

Cardiovascular disease (CVD) is the leading cause of death worldwide,and atherosclerosis is the pathology that causes most of thecardiovascular events. Morphological, structural and functionalalterations of PVAT have been associated with CVD risk factors includingatherosclerosis, hypertension, arterial aneurysm and diabeticvasculopathies. Obesity is a major risk factor for CVD. Like otheradipose tissue, PVAT increases in size and expands in obesity andbecomes dysfunctional, which is characterized by hypoxia, infiltrationof immune cells (monocytes, macrophages, lymphocytes and granulocytes),increased production of pro-inflammatory adipokines, cytokines andchemokines. Interestingly, high-fat diet and hypercholesterolemia evenwithout obesity has been shown to increase inflammation in PVAT. Thisinflammation and dysfunction, propagates to the underlying vessel wall,causing vascular endothelial and smooth muscle cell dysfunction,ultimately contributing to atherosclerosis or vascular diseaseformation.

Thus, selective targeting of the perivascular and periadventitialadipose tissue with the slurries described herein may serve as a therapyfor all the vascular diseases and conditions mentioned herein, includingbut not limited to all peripheral vascular diseases, coronary arterydisease, hypertension, aneurysms, diabetic vasculopathies, arrhythmia,arterial spasm, arterial ulcers, neointimal formation, arterialstiffness, arterial atherosclerosis, arterial aneurysm formation such asaortic aneurysm, arrhythmia, and vasculitis. In addition, there isevidence to link PVAT to the pathogenesis of non-atheroscleroticvascular diseases including neointimal formation, aneurysms, arterialstiffness and vasculitis/vasculitic syndromes.

In general, the slurries described herein may be used to selectivelytarget any vessel in the body that plays a role in vascular disease. Forexample, PVAT adjacent to one or more of the coronary arteries, femoralarteries, carotid arteries, ascending aorta artery, abdominal aorta,thoracic aorta, and iliac arteries may be targeted with a slurryinjection to treat vascular disease. Below selective non-limitingexamples are described to provide further motivation for the use ofslurries in the treatment of vascular diseases. The non-limitingexamples are by no means a limiting list of the applications for whichslurry may be used as a treatment for vascular disease.

Atherosclerosis and Peripheral Vascular Disease

The essential role that PVAT plays in inducing inflammation andatherosclerosis was experimentally tested by transplantingpro-inflammatory adipose tissue to the midperivascular area of commoncarotid arteries, which do not normally develop atherosclerosis andshowing that it results in atherosclerotic lesions in mouse models.Ohman M K, Luo W, Wang H, et al. Perivascular visceral adipose tissueinduces atherosclerosis in apolipoprotein E deficient mice.Atherosclerosis. November 2011; 219(1):33-39. A postpartum study ofatherosclerotic patients found that PVAT mass was positively correlatedwith atherosclerotic plaque size. Verhagen S N, Vink A, van der Graaf Y,Visseren F L. Coronary perivascular adipose tissue characteristics arerelated to atherosclerotic plaque size and composition. A post-mortemstudy. Atherosclerosis. November 2012; 225(1):99-104.

Adipose tissue is an active endocrine and paracrine organ, whichcommunicates with the arterial vessel wall and can influence thedevelopment of atherosclerosis and vascular disease. In the setting ofobesity, adipose tissue produces a variety of inflammatory cytokines (oradipokines) that play an essential role in modulating and propagatingatherogenesis. In particular, adipose tissue located on the surface ofthe heart surrounding large coronary arteries (i.e. epicardialperivascular adipose tissue) has been implicated in the pathogenesis ofcoronary artery disease. Atherosclerotic plaques have been shown tooccur predominantly in areas of the coronary arteries that are encasedin PVAT, and the severity of these lesions are directly correlated tothe volume of epicardial PVAT. Payne G A, Borbouse L, Kumar S, et al.Epicardial perivascular adipose-derived leptin exacerbates coronaryendothelial dysfunction in metabolic syndrome via a protein kinaseC-beta pathway. Arterioscler Thromb Vasc Biol. September 2010;30(9):1711-1717. In addition, the Framingham Heart Study andMulti-Ethnic Study of Atherosclerosis identified epicardial andpericardial adipose volume as independent risk marker for cardiovascularand coronary heath disease, and the epicaridal PVAT volume was thestrongest predictor of coronary artherosclerosis. Greif M, Becker A, vonZiegler F, et al. Pericardial adipose tissue determined by dual sourceCT is a risk factor for coronary atherosclerosis. Arterioscler ThrombVasc Biol. May 2009; 29(5):781-786.

Epicardial adipose tissue (EAT) around coronary arteries may inducevasocrine or paracrine effects on the adjacent arterial wall toinfluence atherosclerotic plaque composition, resulting in thedevelopment of high-risk plaque. Nerlekar N, Brown A J, Muthalaly R G,et al. Association of Epicardial Adipose Tissue and High-Risk PlaqueCharacteristics: A Systematic Review and Meta-Analysis. J Am HeartAssoc. Aug. 23 2017; 6(8). Recently in a swine model, surgical removalof 1.5 cm² of EAT around the coronary artery was shown to arrestedcoronary atherogenesis. McKenney-Drake M L, Rodenbeck S D, Bruning R S,et al. Epicardial Adipose Tissue Removal Potentiates Outward Remodelingand Arrests Coronary Atherogenesis. Ann Thorac Surg. May 2017;103(5):1622-1630. However, surgical removal of epicardial adipose tissueis an invasive procedure with various potential surgery relatedcomplications. The slurries described herein may be used to target andremove epicardial adipose around the coronary arteries using a safe andsimple slurry injection. For example, the slurries described herein maybe used to target epicardial adipose tissue directly surroundingcoronary vessels that have atherosclerotic plaques.

Aneurysms

The most widely studied aneurysms are the abdominal aortic aneurysms(AAA), in which inflammation and cellular composition of arterial wall,perivascular and adventitial fat tissue have been shown to play a majorrole. Inflammatory cell such as macrophages, neutrophils, monocytes,lymphocytes, and inflammatory cytokines such as MCP-1, TNF-alpha, IL-6have all been shown to be increased in the aortic wall of AAA.Abdul-Hussien H, Hanemaaijer R, Kleemann R, Verhaaren B F, van Bockel JH, Lindeman J H. The pathophysiology of abdominal aortic aneurysmgrowth: corresponding and discordant inflammatory and proteolyticprocesses in abdominal aortic and popliteal artery aneurysms. J VascSurg. June 2010; 51(6):1479-1487. These inflammatory cells have alsobeen observed in PVAT and have clearly been shown to increasesusceptibility to AAA formation. Police S B, Thatcher S E, Charnigo R,Daugherty A, Cassis L A. Obesity promotes inflammation in periaorticadipose tissue and angiotensin II-induced abdominal aortic aneurysmformation. Arterioscler Thromb Vasc Biol. October 2009;29(10):1458-1464. More recently it was shown that the abnormalappearance of adipocytes in the vascular wall of aortic aneurysm wasstrongly associated with AAA rupture, in a rat abdominal aortic aneurysmmodel. Kugo H, Zaima N, Tanaka H, et al. Pathological Analysis of theRuptured Vascular Wall of Hypoperfusion-induced Abdominal AorticAneurysm Animal Model. J Oleo Sci. May 1 2017; 66(5):499-506. Theabnormal appearance of adipocytes was also shown in human AAA tissue.Tanaka H, Zaima N, Sasaki T, et al. Imaging Mass Spectrometry Reveals aUnique Distribution of Triglycerides in the Abdominal Aortic AneurysmalWall. J Vasc Res. 2015; 52(2):127-135. Furthermore, aortic aneurysm (AA)is a disease that involves progressive dilation of the aorta due toweakening of the artery vascular wall due degradation of extracellularmatrix collagen and elastin fibers which play an important role inmaintaining the integrity and elasticity of the vascular wall. In thestudy by Kugo, H. et al, it was shown that in the areas of AAA whereabnormal appearance of adipocytes was seen in the vessel wall, there wasincreased MCP-1 protein level, and macrophage infiltration around theadipocytes. Kugo H, Zaima N, Tanaka H, et al. Pathological Analysis ofthe Ruptured Vascular Wall of Hypoperfusion-induced Abdominal AorticAneurysm Animal Model. J Oleo Sci. May 1 2017; 66(5):499-506. Electronmicroscopy showed that the presence of these adipocytes caused decreasedcollagen in the vascular wall, thus leading to the conclusion that inthe vascular wall integrity is decreased in areas around adipocytescompares to that in areas without adipocytes. Id.

In conventional medicine, there is no effective medicine available forinhibiting aneurysm growth or preventing aneurysm rupture. Generally,perivascular and adventitial adipocytes are implicated in many of thecommon vascular arterial diseases, which lead to aneurysm formation.Thus, treating those areas with slurry injection will not only lead toselective targeting and reduction of abnormal adipocytes but also leadto increase collagen formation, which we have shown to happen insubcutaneous adipose tissue.

Hypertension

Hypertension is associated with activation of renin-angiotensin system(RAS) and increased vascular oxidative stress, which has been shown tostart from inflammation within the PVAT, and PVAT/adventitial border.Nosalski R, Guzik T J. Perivascular adipose tissue inflammation invascular disease. Br J Pharmacol. October 2017; 174(20):3496-3513.During the progression of hypertension, immune cells mainly infiltratethe perivascular fat tissue surrounding large and resistance vesselssuch as aorta and mesenteric arteries. PVAT has direct influence on thevasoactive and vasodilatory abilities of the artery. Furthermore thenon-cholinergic and non-adrenergic neural network also is present withinthis adipose layer such that both have an influence on vasomotor tone aswell. Under normal physiology there is a strict orderly equilibrium ofthis periarterial neuro-paracrine system, however in obesity, theseputative vasodilators release and effect are blunted. The end result ofsuch would be an increase vasomotor tone with tendency to developingclinical hypertension. Thus, targeted removal of perivascular fat withslurry injection may be a treatment for hypertension.

Injection Systems for Using Slurry to Treat Vascular Disease

In general, an injection system configured to deliver one or more of theslurries described herein into or around PVAT or periadventitial adiposetissue may include an injection device (e.g., a needle, a catheter, apump, etc.). FIG. 7 illustrates one non-limiting example of an injectionsystem 700 that may be utilized to inject the slurries described hereininto or around a desired tissue region (e.g., PVAT or periadventitialadipose tissue). In the illustrated non-limiting example, the injectionsystem 700 includes a catheter 702 and a flow device 704. In somenon-limiting examples, the flow device 704 may be in the form of a pump.

The catheter 702 may include an outer surface 706 and a central lumen708. The outer surface 706 and the central lumen 708 extend along thecatheter 702 from a proximal end 710 to a distal end 712. A tip 714 maybe arranged at the distal end 712 of the catheter.

In the illustrated non-limiting example, the outer surface 706 of thecatheter may be fenestrated along a predefined axial length thereof. Thefenestrated length of the outer surface 706 may be arranged adjacent tothe distal end 712 of the catheter 702. The fenestrated length of theouter surface 706 may include one or more suction apertures 716 thatextend radially through the outer surface 706. The one or more suctionapertures 716 may provide fluid communication between the desired tissueregion and an outer passageway 718 defined along the interior of thecatheter 702. The outer passageway 718 may be arranged radially betweenthe outer surface 706 and the central lumen 708.

In operation, the flow device 704 may provide a desired volume or flowrate of one of the slurries described herein to the central lumen 708.The slurry may be at a predetermined temperature. The slurry may flowalong the central lumen 708, through the tip 714 and into or around thedesired tissue region, for example, to treat a vascular disease. In someapplications, the delivered slurry may melt, for example, withinminutes, and the melted slurry may be suctioned from the desired tissueregion through the one or more suction apertures 716 and along the outerpassageway 718. In some non-limiting examples, the flow device 704 maybe configured to both provide the slurry to the central lumen 708 andsuction the melted slurry through the outer passageway 718. In somenon-limiting examples, as illustrated in FIG. 8, the flow device 704 mayprovide the slurry to the central lumen 708, and a suction device 720may suction the melted slurry through the one or more suction apertures716 and along the outer passageway 718.

In some non-limiting examples, as illustrated in FIG. 9, the injectionsystem 700 may include a balloon-based catheter 900. The balloon-basedcatheter 900 may include an inflatable balloon 902 arranged at a distalend 904 thereof. The flow device 704 may be configured to provide slurrythrough the balloon-based catheter 900, which inflates the balloon 902and recirculates slurry around the balloon 902. Upon inflation of theballoon 902, the balloon 902 may come into contact with a desired tissueregion (e.g., PVAT or periadventitial adipose tissue).

In some applications, the use of the balloon-based catheter 900 mayallow precooling or augmented cooling. In some applications, theballoon-based catheter 900 may allow colder and non-physiologic slurriesto be used, because the slurry will not come into direct contact withtissue. For example, the slurries may be manufactured with a higherglycerol content (e.g., 40-50%) so the slurries are capable of beingmade colder (e.g., close to −20° C. or colder).

In some non-limiting examples, the use of video assisted thoracoscopysurgery may be leveraged to deliver the slurry to PVAT orperiadventitial adipose tissue. In some non-limiting examples,thoracoscopic surgery with long access needles can be used to deliverslurry to the epicardial adipose tissue around coronary arteries.

WORKING EXAMPLES

Quantitative Model to Illustrate the Behavior of Injected Slurries

Simplifying and reasonable assumptions are made in a quantitative modelto illustrate the behavior of injected slurries, as depicted in FIG.36A.

Heat capacity is an important component of the heat exchange between aslurry and a tissue. The first heat exchange to consider is that of theenergy stored by the heat capacity of slurry and tissue. The energy perunit volume in a medium stored by heat capacity is given by H=TρC, whereH is energy density (cal/cm³), T is temperature (° C.), ρ is density(gm/cm³), and C is specific heat capacity (cal/° C. gm). Assume that ρCis the same for slurry and tissue and water, i.e. ρC=1 cal/gm-° C. Thisassumption is approximately true for all soft tissues except fat, forwhich ρC is lower by about a factor of 2.

Consider a local volume of tissue into which slurry has been introduced.When slurry is introduced with a volume fraction of f_(s) into localtissue, the local tissue occupies a volume fraction of (1−f_(s)). Thestored heat per unit volume of the resulting slurry-tissue mix due toheat capacity of the slurry is H_(s)=f_(s)T_(s)ρC, and the stored heatper unit volume due to heat capacity of the tissue isH_(t)=(1−f_(s))T_(t)ρC. After rapid exchange of the thermal energy dueto heat capacity, a new temperature T_(m) is achieved. Thermal energydue to heat capacity of the mix is given by H_(m)=T_(m)ρC. Conservationof energy in the local heat exchange requires that H_(s)+H_(t)=H_(m).Combining the equations:f _(s) T _(s) ρC+(1−f _(s))T _(t) ρC=T _(m) ρC

Solving for T_(m), the slurry-tissue mix temperature after this initialpart of heat exchange:T _(m) =f _(s) T _(s)+(1−f _(s))T _(t)

Because the temperature of physiological ice slurries is generally closeto 0, this simplifies to:T _(m)=(1−f _(s))T _(t)

The rapid heat exchange upon mixing due to heat capacity alone is thevolume-weighted average of the two starting temperatures. For example,if f_(s)=0, no slurry is added, and T_(m)=T_(t), the starting tissuetemperature. If f_(s)=1, the mix is all slurry, and T_(m)=0. Iff_(s)=0.5, there is a 50%-50% mix of slurry and tissue, and theresultant temperature after mixing is the average of the slurry and thetissue starting temperatures. Typical values of f_(s) for interstitialinjection of a slurry range from about 0.2 to about 0.8, i.e., the mixedslurry-tissue volume may have about 20% to 80% slurry content. Alsoconsider the situation of f_(s)=0.5. If the starting tissue temperatureT_(t) is 37° C., then Tm=18.5° C. after exchange of heat from heatcapacity.

The volume fraction of ice in a physiological slurry in this model isdefined as I_(s), being the volume of ice per unit volume of slurry.Immediately after injection into tissue, the initial volume fraction ofice in the local slurry-tissue mix, is therefore:I _(o) =f _(s) I _(s)

wherein I_(o) is the total amount of ice available for melting, per unitvolume of the slurry-tissue mix.

After the rapid heat exchange from heat capacity, ice in the slurrycomponent of the slurry-tissue mix begins to melt, absorbing heat andcooling the slurry-tissue mix. Ice in the slurry-tissue mix melts untilit is gone, or until an equilibrium temperature is reached, before theperiod of gradual warming by body heat exchange briefly discussed above.In pure water, ice and liquid water can co-exist at equilibriumtemperatures between 0° C. and 4° C. In tissue, there are numeroussolutes that cause freezing point depression, such that ice and waterco-exist over a somewhat lower temperature range, e.g., about −8° C. to0° C. in skin. Lipids in the tissue are in a liquid state at normal bodytemperature. As cooling of the slurry-tissue mix occurs due to icemelting, below a certain temperature lipids can crystallize. In essence,there is a heat exchange between the latent heat of fusion from meltingice, and the latent heat of fusion from lipid crystallization. These twoprocesses proceed in opposite directions (e.g., the water melts, thelipids crystallize) because lipid crystallization occurs at temperaturesconsiderably higher than the freezing point of water. Most animal fatscrystallize at between 10° C. and 15° C., depending on the length andsaturation of the lipid chains in triglyceride molecules. Wax esters andfree fatty acids crystallize at similar temperatures. Polar lipidscrystallize at lower temperatures, for example the phospholipids of cellmembranes can remain somewhat fluid even well below 0° C.

Injected physiological slurries are effective to inhibit pain or itch byaffecting nerve myelin sheath lipids. Lipids of the sheath crystallizewell above 0° C. Effective treatment depends on variables including thestarting tissue temperature T_(t), the ice content of slurry I_(s), theamount and speed of slurry injected to achieve an adequate slurryfraction f_(s) in the slurry-tissue mix, the target lipid content of thetissue L_(t), its crystallization temperature T_(c), and the time forwhich some ice remains in the slurry-tissue mix.

Enthalpy of fusion (also called heat of fusion) describes how muchthermal energy is absorbed (endothermic) or released (exothermic) due tochanging from solid to liquid state. The melting of ice is anendothermic transition requiring a large amount of thermal energy. Forwater, the heat of fusion is 80 cal/gm. The density of ice at 0° C. is0.92, such that the volumetric heat of fusion, H_(ice) (the heat energyneeded to melt a volume of ice) is:H _(ice)=74 cal/cm³

The total heat per unit volume that can be absorbed by melting all ofthe ice in the slurry-tissue mix, Q_(icetotal) is simply its total icecontent multiplied by H_(ice):Q _(icetotal) =f _(s) I _(s) H _(ice)

Typical values as mentioned above for f_(s) range from about 0.2 to 0.8,and the ice content of physiological slurry can be up to about 50%(I_(s)˜0.5). For the approximate maximum of I_(s)=0.5, the range(without limitation) for Q_(icetotal) in the slurry-tissue mix istherefore about 7 to 30 cal/cm³.

The heat of fusion for animal fat lipids ranges from about 30-50 cal/gm.The density of lipids range from about 0.8-0.9 gm/cm³ (e.g., palmitictriglyceride in solid phase is 0.85 gm/cm³). Taking the mean value of 40cal/gm as the heat of fusion, the latent heat per unit volume forcrystallization of lipids is about:H _(lipid)=34 cal/cm³.

Thus, the latent heat for crystallization of lipids is less than half ofthat for melting of ice. Cooling of the slurry-tissue mix proceeds bysome ice melting, until the temperature reaches about 10° C., thetemperature necessary for lipid crystallization to begin. The thermalenergy from consumed by dropping the temperature of the slurry-tissuemix to about 10° C. is given by:Q _(to10° C.)=(T _(m)−10)ρC.

At about that temperature, whatever ice remains from the slurry willmelt, absorbing the energy necessary to crystallize about twice its ownvolume of lipid. If all of the tissue lipid is crystallized, more icewill melt and the temperature will drop below about 10° C., potentiallyinto the approximately −8° C. to 0° C. range at which ice and liquidwater can coexist in tissue. The lipid content of the slurry-tissue mixis therefore another important factor. Defining the lipid content of thetissue f_(tlip), the lipid content of the slurry-tissue mix is:f _(mlip)=(1−f _(s))f _(tlip).

The value of f_(tlip) depends on tissue type. The lipid content of mostsoft tissues ranges from about 5% (most connective tissues) to about 80%(fat), i.e., f_(tlip)=0.05 to 0.8. The energy per unit volume of theslurry-tissue mix that is produced by crystallizating all of the lipidpresent, is:Q _(liptotal) =f _(mlip) H _(lipid)

During the period of latent heat exchange between ice melting and lipidcrystallization in the slurry-tissue mix, ice in the slurry melts untilall of the lipid is crystallized, or until the ice is gone.

The fraction of the lipid in the slurry-tissue mix that crystallizes issimply given by the energy balance:

$f_{lipxtal} = \frac{Q_{icetotal} - Q_{{to}\; 10^{\circ}\;{C.}}}{Q_{icetotal}}$

If (Q_(icetotal)−Q_(to10° C.))<Q_(liptotal), a fraction of the lipidwill crystallize, given above by f_(lipxtal). If(Q_(icetotal)−Q_(to10° C.))=Q_(liptotal), all of the lipid willcrystallize and all of the ice will melt; the temperature will remainnear about 10° C., the phase transition temperature for most animallipids. If (Q_(icetotal)−Q_(to10° C.))>Q_(liptotal), all of the lipidwill crystallize, and the temperature will thereafter decrease belowabout 10° C. until all of the ice is melted or until an equilibriumexists between ice and liquid water in the tissue, i.e., in thetemperature range of about −8° C. to 0° C. The lowest temperaturereached is determined by heat exchange between the residual ice melting,and the heat capacity of the slurry-tissue mix. The lowest temperatureT_(final) can therefore be estimated by equating the latent heat perunit volume absorbed by melting of the residual ice, with the heatassociated with heat capacity of the temperature drop below about 10° C.

The latent heat associated with the residual ice melting after the lipidis crystallized isQ_(iceresidual)=Q_(icetotal)−Q_(to10° C.)−Q_(liptotal), and the amountof residual ice per unit volume is

$I_{residual} = {\frac{Q_{iceresidual}}{H_{ice}}.}$The temperature drop to T_(final) due to residual ice melting can beestimated by: Q_(iceresidual)˜(10−T_(final))ρC, which rearranges to

${\left. T_{final} \right.\sim 10} - {\frac{Q_{iceresidual}}{\rho\; C}.}$

The local heat exchanges modeled above occur over a time scale ofseconds because the slurry is intimately in contact with tissue, bymixing flowing and/or dissecting through the soft tissue duringinterstitial injection. After exchange of the latent heats from meltingice and crystallizing lipids, the temperature of the slurry-tissue mixsettles at about T_(final), then gradually warms due to conduction andconvection. The rate of gradual warming depends therefore on the ratesof conduction and convection. In the absence of blood flow (convection),warming by conduction involves a minimum characteristic time,proportional to the square of the diameter of the local slurry-tissuemix. Typically in soft tissues, the time in seconds for substantialwarming of a region by conduction (to 1/e of a final equilibrium value)is approximately equal to the square of the diameter in millimeters. Forexample, a 10 mm diameter slurry-tissue mix would typically necessitateabout 100 seconds for substantial warming, and a 30 mm diameterslurry-tissue mix would typically necessitate about 900 seconds (i.e.,15 minutes) for substantial warming by conduction. Depending on the icecontent, some ice may remain even after this estimated period ofsubstantial warming. The model presented here is illustrative, notexact. Direct measurement of slurry and tissue temperatures can beperformed. As shown below, such measurements are generally consistentwith this approximate model.

Ex Vivo Human Abdominoplasty Tissue Experiments

Using ex vivo pig tissue and human abdominoplasty tissue samples,Applicant tested the ability of a sterile, cold injectable slurry toreduce the temperature of adipose tissue.

FIG. 10 depicts an ice slurry with a high concentration of small iceparticles that can be easily injected via a 15-19 gauge needle into thesubcutaneous fat tissue.

FIGS. 11A-11C depict the results of injection of an ice slurry intohuman abdominoplasty adipose tissue. Ice crystals 802 are clearlyvisible in the adipose tissue.

These injections of slurry into tissue create an area of localized icecollection in the subcutaneous fat, which can be detected by ultrasoundas depicted in FIG. 12B in contrast to the ultrasound image of humanskin prior to slurry injection in FIG. 12A. This injected ice was indirect contact with the adipose tissue. The injection of the slurry wasable to reduce the temperature of the adipose tissue below 0° C., whichis well below the crystallization temperature of fat. This experimentdemonstrates that heat exchange between slurry and local fat tissue iscapable of lowering the adipose tissue temperature down to a levelsufficient to damage fat tissue and produce local fat loss.

Slurry has many times (typically 5-8 times, depending on the icecontent) the cooling capacity of liquid coolants (such as cold saline)and is, therefore, able to extract much more thermal energy toselectively damage lipid rich tissue such as fat. For example,embodiments of the sterile and biocompatible slurries described hereingenerate a target tissue temperature of −3° C. to −2° C. Damage to thetarget lipid-rich tissue tends to be enhanced when the cooling rate ishigh at least in part because of limited time for various protectivetissue responses. For example, when 20-25 cc of slurry was injected intosubcutaneous fat, tissue temperatures in the −3° C. to −2° C. range wereproduced nearly instantaneously as depicted in FIG. 13.

FIG. 13 depicts the results of injection of slurry into ex vivo humanabdominoplasty specimen. Prior to injection, a 38° C. heating pad wasplaced underneath the specimen to provide constant heat mimicking humancore temperature. The slurry was injected into human fat tissue using a60 ml syringe and 15 gauge needle with the starting fat temperature of23° C. as measured by a thermocouple embedded into the adipose tissue.After slurry injection, the temperature of the adipose tissue decreasedimmediately down to −3° C. As ice melts in the slurry, tissuetemperature in the tissue immediately adjacent to the slurry ismaintained at or below 0° C. until all of the ice has melted. A singleslurry injection was able to maintain it below 0° C. for at least 10-15minutes.

The low temperatures generated by injected slurry causes localized andselective damage to lipid-rich target tissue such as adipose tissue andmyelinated nerves.

In Vivo Swine Experiments

Investigation of Cryolipolysis in Swine

Applicant conducted experiments injecting physiological sterile iceslurry into the subcutaneous fat of live swine. In this controlledstudy, the effects of slurry injections were compared with injection ofmelted slurry and of normal saline at other sites in the same animal.Injections were performed in accordance with an approved animal studyunder general anesthesia.

Applicant generated sterile biocompatible slurry with a temperature atinjection ranging from +1° C. to −3° C. Prior to injection, ultrasoundmeasurements and standardized photographs were obtained of the sitesthat were to be injected in swine. Sites were injected with cold slurry,room temperature melted-slurry (a solution without ice), water, ornormal saline. Another control site was not injected and received briefskin cooling only. A 15 gauge needle was used, however, Applicantconfirmed in other experiments that the same slurry composition isinjectable through a 19 gauge needle.

Sites were injected through the skin only into an area of approximately4 cm×4 cm. Approximately 20 cc of cold or room temperature slurry andsaline control were successfully delivered to the subcutaneous fat ofpredestinated sites.

After the cold slurry was injected, the temperature inside the injectionsite was as cold as −2° C. The duration of cooling (defined as theperiod in which the temperature of the treatment site remains below +5°C.) ranged from approximately 5 to 19 minutes. After injection, the skinsurface overlying all of the injection sites (experimental and control)becomes raised due to the volume added to fat under the skin. Thisreaction subsides rapidly as the slurry or liquids injected graduallydiffuse into surrounding tissues.

At approximately 3 to 4 weeks of follow-up, injection sites demonstratedobvious depression on gross inspection at the sites where cold slurrywas injected as depicted in FIGS. 14B, 16A, and 16B corresponding toloss of subcutaneous fat. In contrast, there was no apparent depressionat the sites where room temperature slurry, water, and saline wereinjected. It is important to note that on gross examination there was nosign of any damage to surrounding skin tissue. In addition, there was nosign of infection or nonspecific damage to the sites.

Ultrasound images of the sites at baseline (FIGS. 15A and 17A) and at 4weeks after the injection (FIGS. 15B and 17B) clearly demonstrateapproximately 40-50% loss of superficial subcutaneous fat tissue only inthe sites injected with cold slurry.

Referring now to FIGS. 23A and 23B, further experiments were conductedon another pig using normal saline plus 10% glycerol slurry, roomtemperature (melted) slurry, both with and without precooling. FIG. 23Adepicts injection sites before injection and FIG. 23B depicts injectionsites 14 days after injection. On gross observation, there is nodifference between the fat loss at pre-cooling plus slurry and slurryalone, indicating that a single injection of slurry deep in thesubcutaneous fat layer, superficial to the muscular fascia is able toinduce highly rapid, effective loss of subcutaneous fat even when thetissue is not pre-cooled.

In sites where the slurry was injected deep into the muscle, Applicanthas not yet observed any muscular abnormalities, depressions or obviouseffect of slurry on muscle tissue. Histologic analysis by Applicant hasalso shown that muscle tissue is not affected by slurry, thus supportingthe hypothesis that only lipid rich tissue is targeted by cryoslurry.

FIG. 24 depicts a graph of cooling at three points. T3 represents thetemperature of adipose tissue inside the pocket of slurry injection. T2represents the temperature of adipose tissue adjacent to the pocket ofslurry injection. T4 represents the temperature of skin adjacent to thepocket of slurry injection.

FIGS. 25A-25D are photographs of injection site 11, which received aninjection of normal slurry with 10% glycerol at −4.1° C. FIGS. 25A and25B depict the site pre-injection while FIGS. 25C and 25D depict theprominent depression 8 weeks post-injection.

The results of these experiments indicate that slurry injected into thesubcutaneous fat leads to dramatic fat loss within 2-3 weeks after oneinjection based on ultrasound images and gross observation.Additionally, IM (intramuscular) injection of slurry did not cause anygross tissue abnormalities. Cryoslurry is safe and effective intargeting lipid rich tissue such as subcutaneous fat even when adjacentto muscle or other non-lipid-rich tissue. No unwanted side effect suchas skin necrosis, muscle necrosis, infection, damage to other cutaneousstructures was observed after injection based on photographs andhistology. Swine was able to tolerate close to 600 ml of slurryinjections into subcutaneous fat without any sign of volume overload orsystemic abnormalities. Moreover, the rate of cooling is very rapid andthe rate and extent of cooling is related to proximity to injectedslurry as depicted in FIG. 24.

Further injections were performed on another swine as summarized inTable 6 below. 1-2 cycles of 30 cc of slurry were injected into thesubcutaneous fat. In order to demonstrate the presence or absence ofindentation of the subcutaneous fat, a ruler was place horizontal to theskin and lit from the bottom. Light passing under (i.e., between theskin surface and straight edge) is indicative of indentation due to fatloss.

TABLE 6 Pig Experiments Slurry FIGS. Site Slurry Composition Temperature29A & 29B 23 6% Hetastarch + Lactated Ringer's −0.8° C. Solution (+25 ccRoom Temp Normal Saline for Thermal Smoothing) 29C & 29D 25 6%Hetastarch + Lactated Ringer's −0.2° C. Solution (+25 cc Room TempNormal (first Saline for Thermal Smoothing) injection) −0.4° C. (secondinjection) 29E & 29F 27 Lactated Ringer's Solution + 10% −3.2° C.Glycerol (5% in Slurry, 5% Pre- Injected) 29G & 29H 28 5% Glycerol +Lactated Ringer's +0.7° C. Solution 29I-29K 29 Normal Saline −0.2° C.

These experiments highlight the role of ions (e.g., potassium,chlorides, magnesium, calcium, and the like) in increasing the coolingpower of slurries as well as the function of two-phase (i.e., ice andliquid) slurries on fat loss. Additionally, thermal smoothing as used atSites 23 and 25 and chemical smoothing in Site 27 improved theflowability of slurry and reduction of fat. Thermal smoothing refers toallowing a slurry to partially melt prior to injection. Ice particles ina slurry freshly made by mechanically pulverizing ice, have variouspolygonal shapes, similar to the gravel produced when rock ispulverized. Such particles tend to interlock, limiting flowability.Partial melting produces more rounded ice particles, and a slurry withgreater flowability for a given particle size and ice content. Othermethods to improve flowability include using smaller ice particles,lower ice content, adding solute and/or surfactants prior to use of theslurry. Both isotonic and hypertonic solutions were shown to be capableof inducing fat loss. Additionally, slurries containing colloidsolutions such as 6% hetastarch in Lactated Ringer's solution arecapable of inducing fat loss as seen in Sites 23 and 25.

Treatment of Pharyngeal Fat with Ice Slurry

At time of necropsy in a swine, Applicant demonstrated that slurry canbe delivered to the parapharyngeal fat pads using ultrasound guidanceand non-invasive injection. Several drops of black india ink (tattooink) were added to a slurry composed of 10% glycerol (by weight) innormal saline. The addition of ink enables visualization of depositionof the slurry. Ultrasound imaging was used to visualize the area to beinjected. FIG. 28A depicts the injection site. FIG. 28B depicts theinjection depth. FIGS. 28C and 28D depict the localization of the slurry(containing ink) within the parapharyngeal fat pads.

Dermal Thickening

FIGS. 34A and 34B provides images of gross biopsies taken at time ofsacrifice three months post-procedure. FIG. 34A is a cross-section oftissue at a site injected with with a cold slurry of normal saline+10%glycerol. FIG. 34B is a cross-section of tissue at a site injected witha room temperature solution of normal saline+10% glycerol. In the sitereceiving cold slurry injection shown in FIG. 34A, dermal thickening of38.1% was noted at the time of sacrifice. In contrast, the sitereceiving a room temperature solution of the same composition as slurryshown in FIG. 34A did not show any change in the thickness of thedermis.

FIGS. 35A and 35B provide images of histology taken at time of sacrificethree months post-procedure and stained with hematoxylin and eosin(H&E). In the site receiving cold slurry injection in FIG. 35A, septalthickening and increase collagen was noted at the time of sacrifice. Incontrast, the untreated site in FIG. 35B demonstrates normal connectivetissue morphology with thin septae and no collagen production noted.FIGS. 37A and 37B provide images of immunohistochemical (IHC) stainingfor type I collagen taken at time of sacrifice three monthspost-procedure. FIGS. 38A and 38B provide images of immunohistochemical(IHC) staining for type III collagen taken at time of sacrifice threemonths post-procedure.

In Vivo Rat Experiments

Safety and Tolerability Experiments

Rats were injected with a variety of slurry formulations detailed inTable 7 to assess safety and tolerability of the slurries. All of theanimals tolerated the injection with no sign of infection, ulceration,necrosis or side effects.

TABLE 7 Slurry Injections Slurry Composition Temp. Amount NormalSaline + 20% Glycerol −5.2° C. 15 cc Normal Saline + 30% Glycerol −6.7°C. 10 cc Normal Saline + 30% Glycerol −7.4° C. 9-10 cc Normal Saline +40% Glycerol −8.2° C. 9-10 cc Normal Saline + 40% Glycerol −10.1° C.9-10 cc 5% PEG in LR (Lactated Ringer's −0.6° C. 10 cc Solution) + 5%Dextrose 5% PEG in LR + 5% Dextrose −0.8° C. 10 cc 5% PEG in LR + 5%Dextrose −0.2° C. 10 cc Room Temp. 5% PEG in LR + 5% Dextrose 8° C. 10cc 5% TWEEN ® 20 in LR + 5% Dextrose −0.6° C. 10 cc 5% TWEEN ® 20 inLR + 5% Dextrose −0.6° C. 10 cc 5% TWEEN ® 20 in LR + 5% Dextrose −0.6°C. 10 cc Room Temp. 5% TWEEN ® 20 in LR + 16.0° C. 8 cc 5% DextroseHetastarch in LR 0.3° C. 10 cc Hetastarch in LR 0.3° C. 10 cc Hetastarchin LR 0.4° C. 10 cc Room Temp. Hetastrach in LR 15.4° C. 10 ccComparison of Efficacy of Slurry Compositions and Temperatures

Adult Sprague-Dawley rats were anesthetized and given a singlesubcutaneous injection of 10 cc of either cold slurry or a roomtemperature fluid of same composition as cold slurry. The injection wasgiven in the area of the left inguinal fat pad as depicted in theencircled area in FIG. 19. The right side was not injected and used ascontrol.

Three slurry compositions were tested in this experiment: (a) 6%hetastarch in lactated Ringer's solution, (b) 5% TWEEN® 20 (polysorbate20) in lactated Ringer's solution plus 5% dextrose and (c) 5%polyethylene glycol (PEG) in lactated Ringer's solution plus 5%dextrose.

Lactated Ringer's solution is a commonly used intravenous fluid that isisotonic. It is composed of 120 mEq sodium ions, 109 mEq chloride ions,28 mEq lactate, 4 mEq potassium ions, and 3 mEq calcium ions. Thus, itdiffers from normal saline in its composition.

FIGS. 20A and 20B depict the result of injections of 6% hetastarch inlactated Ringer's solution at room temperature (+15.4° C.) and coldslurry (+0.3° C.), respectively. FIG. 20C depicts the control (notinjected) side. FIGS. 20D-20G depict tissue surrounding the injectionsite demonstrating no effects on muscle or surrounding tissue.

All photographs were taken 10 days post-injection procedure. Coldhetastarch slurry resulted in disruption of normal fat morphology (asdepicted with a thin arrow). Adipose tissue is highlighted in brackets.Room temperature slurry did not show disruption of fat tissue on grossobservation. Cold slurry did not show gross changes in muscle or skinnear the injected area. Cooling alone through slurry injection causedthe selective disruption of the fat.

FIGS. 21A and 21B depict the result of injections of 5% TWEEN® 20(polysorbate 20) in lactated Ringer's solution plus 5% dextrose at roomtemperature (+16° C.) and cold slurry (−0.6° C.), respectively, in adultSprague-Dawley rats. FIG. 21C depicts the control (not injected) side.FIGS. 21D-21G depict tissue surrounding the injection site demonstratingno effect on muscle or surrounding tissue.

Both room temperature and cold slurry disrupted normal adiposemorphology relative to control as depicted with the dashed arrow.Adipose tissue is highlighted in brackets. Cold slurry resulted ingreater disruption and potentially greater fat loss as depicted with thesolid arrow. It is possible that adipocytes are sensitive to detergentsin general. Cold slurry did not produce gross changes in muscle or skin.

FIGS. 22A and 22B depict the result of injections of 5% polyethyleneglycol (PEG) in lactated Ringer's solution plus 5% dextrose at roomtemperature (+8° C.) and cold slurry (−0.8° C.), respectively, in adultSprague-Dawley rats. FIG. 19C depicts the control (not injected) side.FIGS. 22D-22G depict tissue surrounding the injection site demonstratingno effect on muscle or surrounding tissue.

Both room temperature and cold slurry disrupted normal adiposemorphology as depicted with dashed arrows relative to control. Adiposetissue is highlighted in brackets. PEG acts as a detergent. It ispossible that adipocytes are sensitive to detergents in general. Coldslurry did not show gross changes in muscle or skin. It is possible thatthe single phase liquid also had some cooling effect, as +8° C. is belowthe point of crystallization of lipids (approximately +14° C.).

These experiments show several results. First, slurries that do notcontain glycerol are also capable of disrupting fat. Second, thedisruption of tissue is selective for fat; there were no observed grosschanges in muscle or overlying skin. Third, in the absence of alipolytic additive, only cold slurry is capable of disrupting fat, as acool or room temperature liquid of the same composition as slurry doesnot result in any changes. The results support the theory thatadipocytes may be more sensitive to injury by substances with detergentproperties, as mild disruption of adipose tissue was observed in theroom temperature variant of slurries containing detergent additives.Fifth, the disruption of tissue was most pronounced in the condition ofcold slurry and a detergent additive, providing preliminary evidencethat there may be an additive or synergistic effect of slurry anddetergent.

Treatment of Visceral Fat

An open laparotomy was performed on obese mice. The perigonadal fat, avisceral fat depot, was exposed and in test mice, was cooled with slurrycomposed of normal saline. In control mice, normal saline warmed to +37°C. was placed on the visceral fat. The animals were closed usingstandard surgical technique, and then sacrificed one weekpost-procedure. The histology of the perigonadal visceral fat at time ofsacrifice in the warm saline group is shown in FIGS. 32A and 32C and thehistology at the time of sacrifice in the slurry group is shown FIGS.32B and 32D, respectively. FIGS. 32A and 32C show normal visceral fatmorphology in the warmed normal saline group. However, disruption of fatmorphology is observed in the saline slurry group depicted in FIGS. 32Band 32D, on both gross histology and high magnification, respectively.

Referring now to FIG. 33, five obese mice were taken from a largercohort, anesthetized, weighed, and each administered a 2 ccintraperitoneal injection of slurry. The slurry injected was composed ofperitoneal dialysis solution (DIANEAL® available from BaxterInternational Inc. of Deerfield, Ill.) and 5% glycerol (w/v). Theinjection temperature was around −1.9° C. The mice were sacrificed 1.5weeks post injection. At time of sacrifice, both the treated mice andthe general cohort were weighed. The mice receiving treatment withslurry lost, on average, 7.9% of their body weight. In contrast, thenormal untreated cohort, gained an average of 21.8% of their bodyweight.

Treatment of Sleep Apnea

Referring now to FIGS. 39A-40B, slurry injections were utilized to treatsleep apnea in a mouse model. FIGS. 39A and 39B are magnetic resonance(MR) images depicting the cross-sections of a control mouse trachea andadjacent tissue at a baseline and four week follow-up, respectively.FIGS. 40A and 40B are magnetic resonance (MR) images depicting thecross-sections of a treated mouse trachea and adjacent tissue at abaseline and four week follow-up, respectively. The mouse treated inFIGS. 40A and 40B was injected with slurry at a temperature of −1.9° C.

TABLE 8 Effect of Slurry Injections to Treat Sleep Apnea in Mouse ModelCross-Sectional Area of Trachea Airway Fat Baseline 4-Week Follow-UpChange Change Control 0.0048 0.0029 −39.6% +68.0% Treated 0.0067 0.0066−1.5% +45.5%Treatment of Vascular Diseases

In general, three swine studies were performed to demonstrate thefeasibility, safety, and effectiveness of using injectable slurries totreat vascular diseases. Specifically, the three swine studies targetedpericardial fat, epicardial fat, and thoracic peri-aortic fat. In somenon-limiting examples, the success of the following swine studiesdemonstrates the potential for injectable slurries to be a minimallyinvasive treatment for vascular diseases including, for example,cardiovascular diseases such as coronary artery disease.

First Swine Study

A 66 kg Yorkshire pig was utilized for the study. Pericardial fat,epicardial fat including right atrioventricular (A-V) groove fat pad,and thoracic fat were injected with a slurry having an initialtemperature of −4° C. The pericardial fat was injected with 10 ml ofslurry, the epicardial fat was injected with 10 ml of slurry and 5 ml ofslurry, and the thoracic peri-aortic fat tissue was injected with 5 mlof slurry. A thermocouple was placed in the tissue to measure adiposetissue temperature before and after injection (see, e.g., FIG. 41 forinjection into epicardial fat tissue on a beating swine heart).

FIG. 42 illustrates the results of the injection into the variousadipose tissues before and after the injection of slurry. In all cases,the adipose tissues showed a rapid decrease in temperature followinginjection of the slurry. For example, the pericardial fat temperaturedropped below 0° C. in less than a minute after injection with theslurry, and maintained a temperature generally around 0° C. for betweentwo and three minutes after injection of slurry.

Second Swine Study

A 71 kg Yorkshire pig was utilized for the experiment. Pericardial fatwas exposed using a left thoracotomy approach and was injected with 45ml of slurry having an initial temperature of −5.7° C. In addition,epicardial adipose tissue was injected with 15 ml and 20 ml of slurryhaving an initial temperature of −5° C., and periappendigial fat wasinjected with 30 ml of slurry having an initial temperature of −5° C.The animal was hemodynamically stable throughout all of the variousslurry injections performed during the experiment.

FIG. 43 illustrates the temperature of the pericardial fat tissue beforeand after the 45 ml slurry injection. The pericardial fat temperaturedecreased to −3.9° C. within 1 minute of injection, and maintained atemperature at or below 0° C. for approximately two minutes afterinjection.

Third Swine Study

A 80 kg female Yorkshire pig was utilized for the experiment.Pericardial adipose tissue was exposed and two cycles of slurryinjections of 14 cubic centimeters (cc) each with one minute apart. Theslurry temperature was −4.8° C. at the time of injection. Two metalclips were applied at the injection sites. No hemodynamic instability orany unwanted side effects were noted during and after injection.

In addition, epicardial atrioventricular (A-V) groove fat pad wasinjected. Two injections of 12 cc and 18 cc were performed with theslurry at a temperature of −5.3° C. and −5.2° C., respectively, at thetime of injection. Two clips were applied to the periphery of theinjection site and inferior to the left atrial appendage in the left A-Vgroove (see, e.g., FIG. 44).

During the slurry injections, the animal tolerated the procedure in theoperating room by showing no signs of hemodynamic instability or cardiacarrhythmia. As such, deliver slurry to adipose tissue around the heartwas safe and tolerated. In addition, the slurry injections brought theadipose tissue close to or below 4° C., which is cold enough to removeadipose tissue. Further, histology after the procedures did not show anysign of scaring or adverse effects to the surrounding tissue.

Adipose Tissue Dose Response Experiments

Yorkshire female pigs weighing between 52-85 kg and 3-6 months old wereutilized for the experiments. Slurry and control melted slurry weredirectly injected into subcutaneous fat ˜2 cm deep at various injectionsites. The slurry was comprised of normal saline (0.9% sodium chloride)and 10% glycerol, and was injected through a 15 gauge needle.

Ultrasound imaging was used to measure fat thickness (SonoSite 10; 7.5MHz linear transducer, SonoSite Inc, Bothell, Wash.) at baseline and 8weeks post treatment. Side-lit photographs of skin indentations weretaken at each site to visually demonstrate subcutaneous adipose tissuereduction. Adipose tissue volume loss was obtained by applying Tegaderm™(3M Medical) tightly over the skin indentation sites and surroundingskin, and measuring the volume of water needed to fill the skinindentation.

In general, the volume of ice injected is equal to the slurry volumetimes its volume fraction of ice. The influence of slurry volume andslurry ice content on the amount of fat thickness was evaluated byinjecting multiple sites with two different volumes (15 ml, 30 ml) attwo different ice contents (20% and 40% by volumes, corresponding to−3.5° C. and −4.8° C. slurries, respectively). Comparisons were made ofreduction of fat thickness (see Table 9, below, which shows the effectof physical modification of slurry on the amount of subcutaneous adiposetissue reduction in each treatment group). Gross pathology measurementswere used for the quantification of the thickness of fat loss becausethis method allows for animal growth correction to be included in themeasurements. Sites in Group 1, treated with 30 ml of slurry with 40%ice content, had total of 12 cm³ ice injected and showed the mostreduction in fat thickness (54.5±5.9%). As illustrated in FIG. 45, sitesin Group 2, treated with 15 ml of slurry with 40% ice content), and inGroup 3, treated with 30 ml of slurry with 20% ice content, both with atotal of 6 cm³ ice injected, showed less reduction in fat thickness incomparison to Group 1 (25.3±3.4% vs 16.7±3%; p<0.05 and p<0.001respectively, Ordinary one-way ANOVA followed by Dunnett's multiplecomparison test). There was no significant difference in fat lossbetween Group 2 and 3.

After the experiments, no scarring or damage to tissue surrounding theinjection sites was observed. As illustrated in FIG. 46, the volume offat loss in these dose-response experiments, was 2.6±0.4 cm³ in Group 1sites; 1.0±0.4 cm³ in Group 2 sites; and 0.7±0.3 cm³ in Group 3 sites.Fat volume loss in Group 2 and Group 3 were significantly less thanGroup 1, and there was again no significant difference between Group 2and Group 3 sites. Quantitatively, the volume of adipose tissue loss wasabout one-fifth of the volume of the injected ice content. Thesedose-response studies highlight the importance of total ice volume forthe biologic effect of the slurry, rather than the volume of slurry.Injected ice volume is the major determinant of how much adipose tissuewill be lost, and the dosing results disclosed herein may beextrapolated to calculate a desired ice volume for a predeterminedamount of adipose tissue removal.

TABLE 9 Thickness and Volume Fat Loss Correlated with Volume andFractional Ice Content of the Injected Slurry Group 1 Group 2 Group 3Control Temperature (° C.) −4.8 −4.8 −3.5 22 Volume of Injection 30 1530 30 (ml) Fractional Ice 40 40 20 0 Content % Total Ice Volume 12 6 6 0(cm³) Gross Fat Thickness 54.5 ± 15.5 25.3 ± 6.8 16.7 ± 5.2  2.64 ± 1.9Reduction % Volume of fat loss 2.6 ± 0.9   1 ± 0.8 0.7 ± 0.6   0 ± 0.0(cm³)

EQUIVALENTS

Although preferred embodiments of the invention have been describedusing specific terms, such description is for illustrative purposesonly, and it is to be understood that changes and variations may be madewithout departing from the spirit or scope of the following claims.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications, andother references cited herein are hereby expressly incorporated hereinin their entireties by reference.

We claim:
 1. A method for treating coronary artery disease by reducingpericardial or epicardial adipose tissue, the method comprising:fabricating a sterile ice slurry including water and ice particles;cooling the sterile ice slurry to a predetermined temperature; andinjecting the sterile ice slurry into a desired tissue region, whereinthe desired tissue region includes pericardial or epicardial adiposetissue.
 2. The method of claim 1, wherein the predetermined temperatureis between 0 degrees Celsius and minus 10 degrees Celsius.
 3. The methodof claim 1, wherein the desired tissue region includes epicardial fat orpericardial fat.
 4. The method of claim 1, wherein the desired tissueregion includes pericardial or epicardial adipose tissue surrounding oneor more coronary arteries.
 5. The method of claim 1, wherein the sterileice slurry further comprises a biocompatible surfactant.
 6. The methodof claim 5, wherein the biocompatible surfactant is glycerol.
 7. Themethod of claim 1, wherein injecting the sterile ice slurry into thedesired tissue region comprises: delivering, via a pump, the sterile iceslurry to the desired tissue region; and suctioning melted slurry fromthe desired tissue region.
 8. The method of claim 1, wherein injectingthe sterile ice slurry into the desired tissue region comprises:inserting a catheter having an injection port and a suction port intothe desired tissue region; delivering the sterile ice slurry to thedesired tissue region through the injection port; and suctioning meltedice slurry from the desired tissue region through the suction port. 9.The method of claim 1, wherein injecting the sterile ice slurry into thedesired tissue region comprises: inserting a balloon-based catheter intoor around the desired tissue region; and recirculating the sterile iceslurry into the balloon-based catheter.
 10. The method of claim 1,wherein the ice particles in the sterile ice slurry have a largestcross-sectional diameter of less than 2 millimeters.
 11. The method ofclaim 1, wherein the epicardial or pericardial adipose tissue is cooledfollowing injection of the sterile ice slurry.
 12. The method of claim1, wherein the injection of the sterile ice slurry reduces the volume ofpericardial or epicardial adipose tissue.
 13. The method of claim 1,wherein the injection of the sterile ice slurry reduces levels ofpro-inflammatory cytokines.
 14. The method of claim 11, wherein thetemperature of the epicardial or pericardial adipose tissue is reducedto a temperature between about minus 10° C. and about 0° C.
 15. Themethod of claim 1, wherein injecting the sterile ice slurry into thedesired tissue region comprises injecting the sterile ice slurry througha needle.